Modified cardiolipin and uses therefor

ABSTRACT

Compositions, methods and devices for the detection of anti-lipoidal antibodies and the diagnosis of disease, for example, syphilis, are described. In particular, oxidized cardiolipins, which may be conjugated with a variety of attachment molecules, such as BSA, KLH, biotin, synthetic protein MAPS, IgY, streptavidin, or avidin, are described. Such oxidized cardiolipin, alone or complexed with one or more attachment molecules, are useful to detect anti-lipoidal antibodies in subjects, for example, when used in lateral flow devices. Lateral flow devices are described that permit the detection of anti-lipoidal antibodies and that permit the co-detection of nontreponemal and treponemal antibodies in biological samples.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/737,901, filed Nov. 18, 2005, which is incorporated herein byreference in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made by the National Center for HIV, STD, and TBPrevention, Division of AIDS, STD, and TB Laboratory Research,Laboratory Reference and Research Branch, Centers for Disease Controland Prevention, an agency of the United States Government.

FIELD

This disclosure relates to modified cardiolipin compositions and usestherefor; in particular, to methods of immobilizing modified cardiolipinto a solid support, and related immunoassays (such as ELISA) andimmunoassay devices (such as test strips, flow-through devices, orlateral flow devices), which assays and devices are useful, for example,for detection of anti-lipoidal antibodies and/or diagnosis of disease(such as syphilis).

BACKGROUND

Syphilis is a sexually transmitted disease (STD) caused by thespirochete bacterium Treponema pallidum. Over 100,000 cases of adultsyphilis are reported worldwide each year. In addition, the disease istransmitted congenitally and affects 3000 or more infants annually. Thecourse of syphilis infection spans many years and may lead to a varietyof clinical presentations, which are characterized by four stages.

The primary stage of syphilis infection occurs 10-100 days afterbacterial infection, and is characterized by the appearance of one ormore chancres (red, bloodless, painless ulcers typically less than 1 cmin diameter). The chancres may appear on the genitalia or elsewhere onthe body. A chancre lasts 3-6 weeks and heals without treatment, leavinga small scar. Infected persons are contagious during this stage.

The secondary stage of syphilis infection is characterized by rash-likeskin lesions that can cover part or all of the body. The skin lesionsare generally painless and appear 1-6 months after the onset of theinitial chancre(s). The skin lesions can resemble warts, pustules, orulcers. Left untreated, they heal in 2-12 weeks without scarring. Fever,sore throat, weakness, weight loss, swelling of the lymph nodes, andloss of the eyelashes and/or part of the eyebrows can also occur duringthis stage of infection. In addition, the symptoms may progress tomeningovascular syphilis, which is characterized by inflammation of thecovering of the brain and spinal cord and/or changes in the vasculatureof the brain. Infected persons are also contagious in the secondaryphase.

The next stage of this disease is latent syphilis or the hidden stage.During this stage, the infected person appears to have recovered and isasymptomatic. This stage lasts for life in approximately two thirds ofpersons who are not treated for syphilis. During the first year oflatency, relapses of secondary stage symptoms may occur. Except during arelapse, infected persons are not contagious during this latent stage;however, children born to latently infected mothers within four years ofthe appearance of the primary chancre may contract congenital syphilis.

Tertiary or late syphilis is the final stage of untreated infection.This stage may occur as early as one year after infection or anytimethereafter with 10 to 20 years being most common. Benign syphilis,characterized by lesions called gummas, can occur in the bone, skin, andinternal organs. Death is rare, but severe disfigurement and pain canoccur. Cardiovascular syphilis is characterized by aortic aneurisms aswell as other cardiovascular problems and frequently results in death.Neurologic involvement may occur in the early stages of syphilis as wellas manifest as late stage symptoms. In the late stage disease,neurosyphilis may be asymptomatic or the patient may have severeneurologic problems such as possible dementia, insanity, impairment ofmobility, blindness, deafness, or even death.

The immune response in syphilis involves production of (i) treponemalantibodies, which are specific for T. pallidum antigens, and (ii)anti-lipoidal antibodies, which recognize lipoidal material releasedfrom damaged host cells, lipoprotein-like material and possiblycardiolipin released from the treponemes. The mainstay of syphilisscreening and diagnosis is serological testing for either or both ofthese two types of antibodies.

Tests for anti-lipoidal antibodies (often called “non-treponemal tests”)are typically based on an antigen composed of naturally occurringcardiolipin, cholesterol and lecithin. The widely used non-treponemaltests (e.g., Venereal Disease Research Laboratory (VDRL) test and RapidPlasma Reagin (RPR) test) monitor, either microscopically (e.g., VDRLtest) or macroscopically (e.g., RPR test), the formation of a flocculentcomprised of antigen-antibody complexes. Non-treponemal tests have theadvantage of being widely available, inexpensive and convenient toperform on large numbers of specimens. Moreover, because anti-lipoidalantibody titers decrease with successful treatment for syphilis,eventually disappearing in most patients, while treponemal antibodiestiters remain high for years or even a lifetime, non-treponemal testsare considered the better choice for monitoring treatment or testing forreinfection.

Treponemal tests are based on antigens derived from T. pallidum andinclude the T. pallidum particle agglutination (TP-PA), the fluorescenttreponemal antibody-absorbed test (FTA-ABS) and enzyme immunoassays.Treponemal tests are used primarily to verify reactivity innon-treponemal tests. The treponemal test may also be used to confirm aclinical impression of syphilis in which the non-treponemal test isnonreactive. Treponemal tests are technically more difficult, timeconsuming, and expensive to perform and cannot be used to monitortreatment because the test will remain reactive for years or a lifetimein approximately 85% of persons successfully treated for syphilis.

Each of the above-described antibody tests is performed using a serumsample that is obtained in a clinical setting and sent to a laboratoryfor analysis. Therefore, test results are typically not available forseveral days after the sample is collected. Because of the frequentdifficulty of tracing patients with STDs, the development of a rapid,point-of-care test is needed to aid the clinician in making a judgment,preferably on the day of testing.

Immunoassay devices (such as test strips, flow-through devices, orlateral flow devices), which offer rapid, on-site results, are availableto qualitatively test serum levels of treponemal antibodies (e.g.,DiaSys Corporation; ACON Laboratories, Inc.; Biokit, S. A.; GenixTechnology; Standard Diagnostics; Cortez Diagnostics, Inc.; and PhoenixBio-Tech Corp). However, analogous tests for anti-lipoidal antibodieshave been more difficult to develop at least in part because thehydrophobic antigens of the anti-lipoidal antibodies (e.g., cardiolipin)resist attachment to solid supports, which is one element of animmunoassay device.

According to some experts, syphilis detection would be further aided bya combination of a non-treponemal test and a treponemal test forscreening and diagnostic purposes. This is an approach advocated by theWorld Health Organization, Treponemal Infections, Technical ReportSeries 674, Geneva: WHO, 1982. An easy-to-use, rapid, point-of-care testcapable of concurrently detecting both non-treponemal and treponemalantibodies would help address this long-felt need.

SUMMARY

Efforts to develop non-solution immunoassays for non-treponemal testing(or combined non-treponemal and treponemal testing) have been frustratedby the difficulty of attaching antigens specifically recognized byanti-lipoidal antibodies, such as cardiolipin, to a solid substrate,such as a nitrocellulose strip. The very small size of the cardiolipinmolecule has resulted in poor localization of the molecule on thesubstrate. The nonpolar nature of the fatty acid side chains ofcardiolipin also imparts a high degree of hydrophobicity to the moleculethat makes it difficult to bind cardiolipin to polar surfaces, such asnitrocellulose. Although the size of the molecule could be increased byconjugating the cardiolipin to larger molecules (such as proteins), suchconjugations have resulted in the loss of antigenicity of thecardiolipin.

The present disclosure provides an approach for reliably attachingcardiolipin (or lipoidal antigens comprising cardiolipin modified asdescribed herein, phosphatidylcholine (also referred to as “lecithin”),and cholesterol) to a solid substrate (such as a microporous membrane ormulti-well plate) while maintaining the antigenicity and specificity ofthe antigen for anti-lipoidal antibodies. Using methods describedherein, it is now possible to create cardiolipin-attachment moleculecomplexes, which can be attached to a variety of solid supports. Theability to attach the immunogenic cardiolipin complex in this mannerallows it to be used in non-solution-based immunoassays, such as ELISAsand immunoassay devices for rapid, on-site testing of non-treponemalantibodies. In certain embodiments, disclosed immunoassay devices alsoincorporate treponemal antigens that are recognized by T. pallidum, suchthat the device conveniently concurrently detects both non-treponemaland treponemal antibodies.

In one particular example, fatty acid side-chains of cardiolipin areoxidized to provide at least one terminal carboxyl group, which is thencross-linked to a polypeptide (such as BSA) that is readily attachable(or already attached) to a solid support (such as the permeablesubstrate of a lateral flow strip or a multi-well plate).

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows digital images of steps in the oxidation of cardiolipin.(A) Unmodified cardiolipin in t-butanol; (B) cardiolipin reactionmixture after the addition of sodium m-periodate and potassiumpermanganate; (C) cardiolipin reaction mixture after the addition ofsodium bisulfite; and (D) two-phase solution obtained aftercentrifugation of mixture (C), wherein several oxidized forms ofcardiolipin are predominantly found in the upper, t-butanol phase.

FIG. 2 shows digital images of thin-layer chromatographs of cardiolipinpreparations. (A) Unmodified cardiolipin in t-butanol (see FIG. 1A); (B)an oxidized cardiolipin preparation; (D1) the upper, t-butanol phasefollowing a cardiolipin oxidation reaction (see FIG. 1D); and (D2) thelower, aqueous phase following a cardiolipin oxidation reaction (seeFIG. 1D). The direction of migration of each sample is shown by anarrow.

FIG. 3 shows a schematic oxidation reaction of an exemplary cardiolipinmolecule that contains four 18-carbon, di-unsaturated fatty acid chains.The illustrated cardiolipin reaction products have either one or fouroxidized fatty acid side chains. In practice, the oxidation reactionproducts would also include oxidized cardiolipin species where any twoor three of the fatty acid chains were oxidized to carboxyl groups.

FIG. 4 shows a schematic reaction useful for covalently linking oxidizedcardiolipin to amine-containing attachment molecules, such as a protein.In the illustrated example, the attachment molecule is BSA.

FIG. 5 shows a digital image of a dot blot assay used to determine theantigenicity of an oxidized cardiolipin-BSA conjugate (“Preparation 5”in Table 2).

FIG. 6 shows a digital image of five different physical embodiments oflateral flow devices that could be used with the disclosed methods. Thedevice embodiments shown in (A), (B) and (E) are configured so that eachmay be dipped into, or partially submerged in, the sample or asample-containing solution. The device embodiments shown in (C) and (D)are configured so as to receive a volume of the sample (or asample-containing solution) dropwise into a sample introduction port.

FIG. 7 is a perspective view of a physical embodiment of a lateral flowdevice, with a portion of the housing broken away to show the basiccomponents of the device and their relationship to each other.

FIG. 8 shows a digital image of a thin-layer chromatograph of anoxidized cardiolipin-lecithin mixture. (A) A mixture of unmodifiedcardiolipin and unmodified lecithin in t-butanol; (B) an oxidizedcardiolipin/lecithin mixture; (C) the upper, t-butanol phase followingthe oxidation of a cardiolipin/lecithin mixture; and (D) the lower,aqueous phase following the oxidation of a cardiolipin/lecithin mixture.The direction of migration of each sample is shown by an arrow.

FIG. 9 shows the chemical structures of four representative naturallyoccurring phosphatidylcholine (lecithin) molecules. ForL-α-phosphatidylcholine (heart, bovine), the molecular formula isC₄₂H₈₀NO₈P, the molecular weight is 758.07, the molecular weight(isotope) is 757.562157, and the percent composition is C 66.55% H10.64% N 1.85% O 16.88% P 4.09%. For L-α-phosphatidylcholine,plasmalogen (heart, bovine), the molecular formula is C₄₂H₈₀NO₇P, themolecular weight is 742.07, the molecular weight (isotope) is741.567242, and the percent composition is C 67.98% H 10.87% N 1.89% O15.09% P 4.17%. For L-α-phosphatidylcholine, ether (heart, bovine), themolecular formula is C₄₂H₈₂NO₇P, the molecular weight is 744.09, themolecular weight (isotope) is 743.582893, and the percent composition isC 67.80% H 11.11% N 1.88% O 15.05% P 4.16%.

FIG. 10 shows a digital image of a dot blot assay used to determine theantigenicity of six oxidized cardiolipin/lecithin-BSA or -KLHconjugates. The amounts of the respective antigens applied to each dotin the assay are given in the table below the blot.

DETAILED DESCRIPTION I. Introduction

As shown in FIG. 3, cardiolipin includes a central immunogenic glycerolmoiety with fatty acid side chains. This specification discloses methodsfor preparing oxidized cardiolipin that is capable of linking to apolypeptide for attachment to a substrate, while retaining the abilityof the central glycerol moiety to be immunoreactive with anti-lipoidalantibodies. In disclosed embodiments, the cardiolipin is reacted with aperiodate salt (such as sodium m-periodate) and a permanganate salt(such as potassium permanganate) to oxidize at least one of the fattyacid side chains of the cardiolipin to provide terminal carboxyl groupson one or more of the side chains, then a reducing agent (such as abisulfite salt) is added to quench oxidation of the cardiolipin and toreduce a β-ketone formed in the central glycerol moeity to a β-hydroxylgroup so as to retain immunogenicity of the central glycerol moiety. Indisclosed embodiments, the cardiolipin is reacted with the periodatesalt before the cardiolipin is reacted with the permanganate salt. Inparticular embodiments, the oxidation reaction with the cardiolipinoccurs in an alcohol solvent, under an argon atmosphere. Examples ofsuitable alcohol solvents are one or more of t-butanol, ethanol,propanol, or methanol. The molar ratio of sodium m-periodate tocardiolipin is about 4:1 to about 5:1, and the molar ratio of potassiumpermanganate to cardiolipin is about 0.5:1 to about 1:1. In someexamples, the bisulfite salt is sodium bisulfite.

The carboxyl groups of the oxidized cardiolipin may be activated toprepare them for covalently attaching a polypeptide carrier to at leastone of the carboxyl groups. Activating the carboxyl groups is achievedin particular examples by reacting the oxidized cardiolipin with acarbodiimide, such as 1-ethyl-3-(3-dimethylamino propyl)carbodiimide(EDC) and a succinimide such as N-hydroxysuccinimide (NHS). Apolypeptide (such as, bovine serum albumin (BSA), keyhole limpethemocyanin (KLH), or avidin), or biotin, is then covalently attached toat least one of the activated carboxyl groups.

Also disclosed herein are methods for preparing oxidized cardiolipinthat is immunoreactive with anti-lipoidal antibodies, and capable oflinking to a polypeptide for attachment to a substrate. Such methodsinclude reacting the cardiolipin with sodium m-periodate and potassiumpermanganate in a t-butanol solvent under an argon atmosphere to oxidizethe cardiolipin and provide terminal carboxyl groups on one or more ofthe side chains. Sodium bisulfite in aqueous solution is then added tothe cardiolipin suspension to quench oxidation of the cardiolipin and toreduce a β-ketone formed in the central glycerol moeity to a β-hydroxylgroup so as to retain immunogenicity of the central glycerol moiety. Theterminal carboxyl groups of the oxidized cardiolipin are then activatedby reacting the oxidized cardiolipin with EDC and then NHS, followed bycovalently conjugating a protein carrier to at least one of the carboxylgroups.

The oxidized cardiolipins conjugated to polypeptide carriers can then beused, in one application, to make a lateral flow chromatography deviceby attaching the cardiolipin-polypeptide conjugate to a lateral flowsubstrate, such as a nitrocellulose strip. The ability to provide thepolypeptide-conjugated cardiolipin permits the cardiolipin to bereliably attached to a solid substrate. Such substrates can also includea treponemal antigen that reacts with antibodies to T. pallidum. Thepresence of both a cardiolipin-polypeptide conjugate and a treponemalantigen on (or in) the same solid support conveniently allows rapidclinical testing for both anti-lipoidal and anti-treponemal antibodiesin the same biological specimen.

Also disclosed herein are immunoassay devices (such as lateral flow orflow-through devices) for determining the presence and/or amount of ananti-lipoidal antibody in a fluid sample. These devices typicallyinclude a sample application area and a separate cardiolipin capturearea in which an immobilized cardiolipin-polypeptide conjugate isprovided which conjugate has a specific binding affinity for amobile-phase anti-lipoidal antibody. Any liquid (such as a fluidbiological sample) applied in the sample application area flows along apath of flow from the sample application area to the cardiolipin capturearea. The path of flow may continue to a downstream absorbent padassociated with the immunoassay device, which acts, at least in part, asa liquid reservoir. Formation of a complex between the anti-lipoidalantibody and the immobilized oxidized cardiolipin can be detected todetermine the presence and/or amount of the anti-lipoidal antibody in afluid sample.

In some embodiments of a lateral flow device, a conjugate pad is placedin the path of flow from the sample application area to the cardiolipincapture area. The conjugate pad includes a mobile or mobilizabledetector reagent for an anti-lipoidal antibody, such that flow of liquidthrough the pad moves the detector reagent to the cardiolipin capturearea. Formation of a complex including the detector reagent,anti-lipoidal antibody (analyte), and immobilized cardiolipin provides avisible or otherwise detectable indicator of the presence of theanti-lipoidal antibody in a biological specimen. In alternativeembodiments (including lateral flow or flow-through devices), thedetector reagent is not supplied in a conjugate pad, but is insteadapplied to the substrate or sample, for example from a developer bottle.

Examples of detector reagents include labeled oxidized cardiolipin orlabeled anti-human antibody, in which the label is one or more of anenzyme, colloidal gold particle, colored latex particle,protein-adsorbed silver particle, protein-adsorbed iron particle,protein-adsorbed copper particle, protein-adsorbed selenium particle,protein-adsorbed sulfur particle, protein-adsorbed tellurium particle,protein-adsorbed carbon particle, or protein-coupled dye sac.

Certain immunoassay device embodiments (such as lateral flow orflow-through devices) also include a treponemal capture area in the flowpath from the sample application area. Such treponemal capture area mayinclude, for example, an immobilized treponemal antigen having aspecific binding affinity for a mobile phase anti-T. pallidum antibodyor an immobilized anti-T. pallidum antibody having a specific bindingaffinity for a mobile phase T. pallidum organism or T. pallidum antigen.A lateral flow device may also have a mobile or mobilizable detectorreagent specific for the treponemal antibody or antigen in the conjugatepad. A detector reagent for the treponemal antibody or antigen may be inthe same or a different pad than the detector reagent for theanti-lipoidal antibody. In particular embodiments, a detector reagentspecific for an anti-T. pallidum antibody comprises labeled (e.g.,gold-conjugated) Protein A, labeled (e.g., gold-conjugated) Protein G,or labeled (e.g., gold-conjugated) anti-human antibody. In otherembodiments, detector reagent for a mobile treponemal antigen can be alabeled (e.g., gold-conjugated) anti-treponemal antigen antibody.

The disclosed immunoassay devices can be used in methods for diagnosingsyphilis in a subject by analyzing a biological sample from the subject,by applying the biological sample to the device and detecting formationof a complex among the anti-lipoidal antibody, the oxidized cardiolipin,and a detector reagent in the capture area. Detection of the formationof the complex in the capture area detects an anti-lipoidal antibodyassociated with infection with syphilis. In those embodiments in whichthe device includes a conjugate pad in the path of flow from the sampleapplication area to the cardiolipin capture area, the detected complexincludes the mobile or mobilizable detector reagent. In otherembodiments in which the detector reagent is applied to the device froman external source, the detected complex includes the externally applieddetector.

In particularly advantageous embodiments of the method, the lateral flowdevice is capable of detecting both the anti-lipoidal antibodies (thatare an indicator of active infection) and anti-treponemal antibodies(that verify reactivity of the non-treponemal test). In thoseembodiments of the device which include the treponemal antigen, themethod includes detecting formation of a complex between the anti-T.pallidum antibody, the immobilized treponemal antigen, and a detectorreagent in the capture area. As with the detector reagent used for thecardiolipin, the detector reagent for the treponemal antigen can beprovided on the device or applied from an external source.

Also disclosed herein are kits for the diagnosis of syphilis. These kitsinclude a disclosed immunoassay device and instructions for applying thebiological sample to the sample application area or the device. The kitmay also include instructions for interpreting results of the test.

The disclosed immunoassay devices can be also used in methods fordiagnosing lupus in a subject by analyzing a biological sample from thesubject, by applying the biological sample to the device and detectingformation of a complex among the anti-lipoidal antibody, the oxidizedcardiolipin, and a detector reagent in the capture area. Detection ofthe formation of the complex in the capture area detects ananti-lipoidal antibody associated with lupus. In some instances, one ormore co-factors (such as 2-glycoprotein I) are present (such as added toa sample) for the detection of lupus

II. Abbreviations and Terms

BSA Bovine serum albumin

EDC 1-ethyl-3-(3-dimethylamino propyl)carbodiimide

ELISA Enzyme-Linked Immunosorbent Assay

HPLC High pressure liquid chromatograph

KLH Keyhole limpet hemocyanin

LFD Lateral flow device

MES N-morpholinoethane sulfonic acid

NHS N-hydroxysulfosuccinimide

NMR Nuclear magnetic resonance

PEG Polyethylene glycol

PVA Polyvinyl alcohol

PVP Polyvinyl pyrrolidone

SDS Sodium dodecyl sulfate

TLC Thin layer chromatography

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

Activating carboxyl groups: Activation of a carboxyl group refers to thereaction of a carboxyl group with an agent (or agents), such as acarbodiimide (e.g., 1-ethyl-3-(3-dimethylamino propyl) carbodiimide), toform a nucleophile-reactive derivative, such as an O-urea derivative.The nucleophile-reactive derivative is readily reactive withnucleophiles, and can be used to form (i) ether links with alcoholgroups, (ii) ester links with acid and alcohols or phenols, and (iii)peptide bonds with acid and amines.

A catalytic auxiliary nucleophile, such as 1-hydroxybenzotriazole,N-hydroxysuccinimide, N-hydroxysulfosuccinimide, andN-hydroxy-5-norbene-endo-2,3-dicarboxamide may be used to assistcarbodiimide-mediated activation of carboxyl groups to reduce possibleside reactions, including racemisation, and to increase reaction ratewhen using active esters.

For example, a terminal carboxyl group of one or more fatty acid sidechains of oxidized cardiolipin may be activated by reaction with1-ethyl-3-(3-dimethylamino propyl)carbodiimide followed, in someinstances, by reaction with a catalytic auxiliary nucleophile, such asN-hydroxysuccinimide.

Analyte: An atom, molecule, group of molecules or compound of natural orsynthetic origin (e.g., drug, hormone, enzyme, growth factor antigen,antibody, hapten, lectin, apoprotein, cofactor) sought to be detected ormeasured that is capable of binding specifically to cardiolipinembodiments described herein. Analytes may include, but are not limitedto antibodies, drugs, hormones, antigens, haptens, lectins, apoproteins,or cofactors. In some embodiments, the analyte includes antibodies, suchas anti-lipoidal or anti-cardiolipin antibodies, produced in response toinfection by T. pallidum. In other embodiments, the analyte includesanti-lipoidal antibodies produced in response to any of (i) anautoimmune disease, such as lupus, (ii) various venous and arterialthrombotic disorders, including cerebral infarction, (iii) deep venousthrombosis, (iv) thrombocytopenia, (v) pulmonary embolism, or (vi)recurrent fetal loss with placental infarction.

Antibody: A protein consisting of one or more polypeptides substantiallyencoded by immunoglobulin genes or fragments of immunoglobulin genes.The recognized immunoglobulin genes include the kappa, lambda, alpha,gamma, delta, epsilon and mu constant region genes, as well as themyriad of immunoglobulin variable region genes. Light chains areclassified as either kappa or lambda. Heavy chains are classified asgamma, mu, alpha, delta, or epsilon, which in turn define theimmunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and “variable heavy chain” (V_(H)) refer, respectively, to theselight and heavy chains.

Antibodies are naturally produced in plants and animals in response toantigens presented to the immune system. Naturally produced antibodiesmay be found, for example, in the serum of an animal. For example, aperson infected with T. pallidum will produce antibodies at leastagainst T. pallidum antigens and antibodies (i.e., anti-lipoidalantibodies) against lipoidal material that results from the treponemalinfection, for example, from host cells damaged by the infection.

Antibodies may exist as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases or by recombinant DNA methods. For example, pepsin digests anantibody below the disulfide linkages in the hinge region to produceF(ab)′₂, a dimer of Fab which itself is a light chain joined toV_(H)-C_(H) 1 by a disulfide bond. The F(ab)′₂ may be reduced under mildconditions to break the disulfide linkage in the hinge region therebyconverting the F(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer isessentially an Fab with part of the hinge region (see FundamentalImmunology, W. E. Paul, ed., Raven Press, N.Y., 1993). While certainantibody fragments are defined in terms of the digestion of an intactantibody, it will be appreciated that Fab′ fragments or other antibodyfragments may be synthesized de novo either chemically or by utilizingrecombinant DNA methodology. Thus, the term antibody as used herein alsoincludes antibody fragments either produced by the modification of wholeantibodies or synthesized de novo using recombinant DNA methodologies.

Anti-lipoidal antibodies for use in some embodiments of the methods anddevices disclosed herein can be of any derivation, but most often willbe found in the serum of a subject.

Antibodies may be monoclonal or polyclonal. Merely by way of example,monoclonal antibodies can be prepared from murine hybridomas accordingto the classical method of Kohler and Milstein (Nature 256:495-497,1975) or derivative methods thereof. Briefly, a mouse is repetitivelyinoculated with a few micrograms of the selected analyte compound (or afragment thereof) over a period of a few weeks. In some instances, itwill be beneficial to use an adjuvant or a carrier molecule to increasethe immunogenicity and/or stability of the analyte in the animal system.The mouse is then sacrificed, and the antibody-producing cells of thespleen isolated. The spleen cells are fused by means of polyethyleneglycol with mouse myeloma cells, and the excess un-fused cells destroyedby growth of the system on selective media comprising aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as ELISA, as originally described byEngvall (Meth. Enzymol. 70:419-439, 1980), and derivative methodsthereof. Selected positive clones can be expanded and their monoclonalantibody product harvested for use. Detailed procedures for monoclonalantibody production are described in Harlow and Lane (Antibodies, ALaboratory Manual, CSHL, New York, 1988).

Antigen: A chemical or biochemical compound, composition, structure,determinant, antigen or portion thereof that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes.

Anti-lipoidal antibody: An antibody (such as IgM or IgG) produced by theimmune system of a subject (such as a human) in response to lipoidalantigens present in a disease state, such as an infection. For example,this term contemplates anti-lipoidal antibodies produced in response tolipoidal material released from host cells as a consequence of T.pallidum infection, and lipoprotein-like material and possiblycardiolipin released from treponemes. Anti-lipoidal antibodies may alsobe produced in response to nontreponemal diseases in which lipoidalantigens are also present, including, for example (i) an autoimmunedisease, such as lupus (Harris et al., Clin. Rheum. Dis., 11:591-609,1985), (ii) various venous and arterial thrombotic disorders, includingcerebral infarction (Harris et al., Clin. Exp. Rheumatol., 2:47-51,1984), (iii) deep venous thrombosis (Mueh et al., Ann. Intern. Med.,92:156-159, 1980), (iv) thrombocytopenia (Harris et al., Clin. Rheum.Dis., 11:591-609, 1985), (v) pulmonary embolism (Anderson and Ali, Ann.Rheum. Dis., 43:760-763, 1984), or (vi) recurrent fetal loss withplacental infarction (Derue et al., J. Obstet. Gynaecol., 5:207-209,1985).

Cardiolipin is one commonly known specific binding partner ofanti-lipoidal antibodies that are formed in response to disease states,such as infection by T. pallidum. Naturally occurring cardiolipin hastraditionally been used, often in combination with cholesterol andlecithin, in flocculation tests to detect anti-lipoidal antibodies, forexample, in the serum of T. pallidum-infected patients.

Argon atmosphere: An atmosphere substantially comprising argon gas,which is created for the purposes of performing a chemical reaction. Anargon atmosphere may be created in any closed vessel useful forperforming chemical reactions by flushing the reaction vessel with argongas to displace the atmosphere then-existing inside the vessel, andthereafter maintaining a flow of argon gas sufficient to maintain anatmosphere inside the reaction vessel substantially comprising argongas. An atmosphere substantially comprises argon gas when the atmosphereis at least about 25% argon, at least about 30% argon, at least about40% argon, at least about 50% argon, at least about 60% argon, at leastabout 70% argon, at least about 75% argon, at least about 80% argon, atleast about 85% argon, at least about 90% argon, at least about 92%argon, at least about 95% argon, at least about 98% argon, or at leastabout 99% argon.

Attachment molecule or protein: Any polypeptide or other molecule (e.g.,atom, molecule, group of molecules, protein, polymer, or compound ofnatural or synthetic origin) that can be directly or indirectly linkedto cardiolipin as described herein to facilitate attachment of thecardiolipin to a solid support. In some embodiments, the attachmentmolecule-cardiolipin linkage is formed via carboxyl groups in oxidizedcardiolipin, and/or optionally utilizes a linking group.

Suitable attachment molecules include, but are not limited topolypeptides, proteins, or peptides such as albumin, hemocyanin,thyroglobulin and derivatives thereof, particularly bovine serum albumin(BSA), synthetic protein MAPS, IgY, streptavidin, avidin, and keyholelimpet hemocyanin (KLH). Other polypeptide-derived or non-proteinderived substances are known to those skilled in the art. An attachmentmolecule typically has a molecular weight of at least 50,000 daltons,preferably greater than 60,000 daltons. Attachment molecules oftencontain a reactive group to facilitate covalent conjugation to theoxidized cardiolipin. The amine groups of amino acids are often used inthis manner. Attachment molecules lacking such groups can often bereacted with an appropriate chemical to produce them.

Binding affinity: A term that refers to the strength of binding of onemolecule to another at a site on the molecule. If a particular moleculewill bind to or specifically associate with another particular molecule,these two molecules are said to exhibit binding affinity for each other.Binding affinity is related to the association constant and dissociationconstant for a pair of molecules, but it is not critical to theinvention that these constants be measured or determined. Rather,affinities as used herein to describe interactions between molecules ofthe described methods and devices are generally apparent affinities(unless otherwise specified) observed in empirical studies, which can beused to compare the relative strength with which one molecule (e.g., anantibody or other specific binding partner) will bind two othermolecules (e.g., an analyte and an analyte-tracer conjugate). Theconcepts of binding affinity, association constant, and dissociationconstant are well known.

Binding domain: The molecular structure associated with that portion ofa receptor that binds ligand. More particularly, the binding domain mayrefer to a polypeptide, natural or synthetic, or nucleic acid encodingsuch a polypeptide, the amino acid sequence of which represents aspecific (binding domain) region of a protein, which either alone or incombination with other domains, exhibits binding characteristics thatare the same or similar to those of a desired ligand/receptor bindingpair. Neither the specific sequences nor the specific boundaries of suchdomains are critical, as long as binding activity is exhibited.Likewise, used in this context, binding characteristics necessarilyincludes a range of affinities, avidities and specificities, andcombinations thereof, as long as binding activity is exhibited.

Biological sample: Any sample that may be obtained directly orindirectly from a subject, including whole blood, plasma, serum, tears,mucus, saliva, urine, pleural fluid, spinal fluid, gastric fluid, sweat,semen, vaginal secretion, sputum, fluid from ulcers and/or other surfaceeruptions, blisters, abscesses, and/or extracts of tissues, cells ororgans. The biological sample may also be a laboratory research samplesuch as a cell culture supernatant. The sample is collected or obtainedusing methods well known to those skilled in the art.

Carbodiimide: A compound of the structure R₁—N═C═N—R₂, where R₁ and R₂are independently hydrogen or hydrocarbyl groups. Specific examplesinclude HN═C═NH, 1-ethyl-3-(3-dimethylamino propyl)carbodiimide;N,N′-dicyclohexyl-carbodiimide; and diisopropylcarbodiimide.

Cardiolipin: Includes both natural cardiolipin and any modifiedcardiolipin that has structural similarity to naturally occurringcardiolipin, and which is capable of specifically binding to ananti-lipoidal antibody.

Cardiolipin capture area: A capture area wherein cardiolipin isimmobilized as the capture reagent.

Capture reagent: An unlabeled specific binding partner that is specificfor (i) an analyte, as in a sandwich assay, or (ii) a detector reagentor an analyte, as in a competitive assay, or for (iii) an ancillaryspecific binding partner, which itself is specific for the analyte, asin an indirect assay. As used herein, an “ancillary specific bindingpartner” is a specific binding partner that binds to the specificbinding partner of an analyte. For example, an ancillary specificbinding partner may include an antibody specific for another antibody,for example, goat anti-human antibody. A “capture area” is a region of alateral flow device where the capture reagent is immobilized. A lateralflow device may have more than one capture area, for example, a “primarycapture area,” a “secondary capture area,” and so on. Often a differentcapture reagent will be immobilized in the primary, secondary, or othercapture areas. Multiple capture areas may have any orientation withrespect to each other on the lateral flow substrate; for example, aprimary capture area may be distal or proximal to a secondary (or other)capture area and vice versa. Alternatively, a primary capture area and asecondary (or other) capture area may be oriented perpendicularly toeach other such that the two (or more) capture areas form a cross or aplus sign or other symbol.

Conjugate: When used in the verb form, the term “conjugate” means thecovalent coupling of one molecule (e.g., oxidized cardiolipin) toanother molecule (e.g., a protein). Such coupling may be achieved bychemical means, either with or without the use of a linking group. Whenused in the noun form, the term “conjugate” means a coupled molecularcomplex formed by conjugation.

Detecting or Detection: Refers to quantitatively or quantitativelydetermining the presence of the analyte(s) under investigation (e.g.,anti-lipoidal antibodies and/or anti-T. pallidum antibodies). “DetectingFormation of a Complex” refers to detecting a complex comprising adetector reagent by any method suitable for observing the particularlabel associated with the detector reagent; for instance, visualobservation of a colored (or otherwise visible) label, measurement orvisual detection of a fluorescent, chemiluminescent or radioactivelabel.

Detector reagent (or Detection reagent): A specific binding partner thatis conjugated to a label. Detector reagents include, for example,labeled analyte-specific binding members (such as gold-conjugated,oxidized cardiolipin-attachment molecule complexes), or labeledancillary specific binding members (such as enzyme-conjugate, goatanti-human antibodies).

Epitope (or antigenic determinant): A site on the surface of an antigenmolecule to which a single antibody molecule binds; generally an antigenhas several or many different antigenic determinants and reacts withantibodies of many different specificities.

Immunogenicity: The property of being able to evoke an immune responsewithin an organism. For example, oxidized cardiolipin retainsimmunogenicity when an anti-lipoidal antibody has the ability to bind anepitope present in oxidized cardiolipin.

Label: Any molecule or composition bound to an analyte, analyte analog,detector reagent, or binding partner that is detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Examples of labels, including enzymes,colloidal gold particles, colored latex particles, have been disclosed(U.S. Pat. Nos. 4,275,149; 4,313,734; 4,373,932; and 4,954,452, eachincorporated by reference herein). Additional examples of useful labelsinclude, without limitation, radioactive isotopes, co-factors, ligands,chemiluminescent or fluorescent agents, protein-adsorbed silverparticles, protein-adsorbed iron particles, protein-adsorbed copperparticles, protein-adsorbed selenium particles, protein-adsorbed sulfurparticles, protein-adsorbed tellurium particles, protein-adsorbed carbonparticles, and protein-coupled dye sacs. The attachment of a compound(e.g., a detector reagent) to a label can be through covalent bonds,adsorption processes, hydrophobic and/or electrostatic bonds, as inchelates and the like, or combinations of these bonds and interactionsand/or may involve a linking group.

Lateral flow device: An analytical device in the form of a test stripused in lateral flow chromatography, in which a test sample fluid,suspected of containing an analyte, flows (for example by capillaryaction) through the strip (which is frequently made of bibulousmaterials such as paper, nitrocellulose, and cellulose). The test fluidand any suspended analyte can flow along the strip to a detection zonein which the analyte (if present) interacts with a detection agent toindicate a presence, absence and/or quantity of the analyte.

Numerous lateral flow analytical devices have been disclosed, andinclude those shown in U.S. Pat. Nos. 4,313,734; 4,435,504; 4,775,636;4,703,017; 4,740,468; 4,806,311; 4,806,312; 4,861,711; 4,855,240;4,857,453; 4,943,522; 4,945,042; 4,496,654; 5,001,049; 5,075,078;5,126,241; 5,451,504; 5,424,193; 5,712,172; 6,555,390; and 6,368,876; EP0810436; and WO 92/12428; WO 94/01775; WO 95/16207; and WO 97/06439,each of which is incorporated by reference.

Many lateral flow devices are one-step lateral flow assays in which abiological fluid is placed in a sample area on a bibulous strip (though,non-bibulous materials can be used, and rendered bibulous by applying asurfactant to the material), and allowed to migrate along the stripuntil the liquid comes into contact with a specific binding partner thatinteracts with an analyte in the liquid. Once the analyte interacts withthe binding partner, a signal (such as a fluorescent or otherwisevisible dye) indicates that the interaction has occurred. Multiplediscrete binding partners can be placed on the strip (for example inparallel lines) to detect multiple analytes in the liquid. The teststrips can also incorporate control indicators, which provide a signalthat the test has adequately been performed, even if a positive signalindicating the presence (or absence) of an analyte is not seen on thestrip.

Lecithin (a.k.a., phosphatidylcholine): Lecithin is the common name forphosphatidylcholine. Phosphatidylcholine is a glycerophospholipid, whichis usually the most abundant phospholipid in animal and plants. It is akey building block of membrane bilayers, and is also the principalphospholipid circulating in the plasma. Phosphatidylcholine is a neutralor zwitterionic phospholipid over a pH range from strongly acid tostrongly alkaline. Phosphatidylcholine contains two fatty acid sidechains, which may have variable chemical structures in nature and asmodified synthetically. The chemical structures of four representativenaturally occurring phosphatidylcholine molecules are shown in FIG. 9.

As used herein, the term “lecithin” includes both naturally occurringlecithins and any modified synthetic lecithin that has structuralsimilarity to naturally occurring lecithin. For example, the fatty acidsof synthetic lecithins may include the following:

Carbon Number 1-Acyl 2-Acyl 14:0-16:0 Myristoyl Palmitoyl 14:0-18:0Myristoyl Stearoyl 16:0-14:0 Palmitoyl Myristoyl 16:0-18:0 PalmitoylStearoyl 16:0-18:1 Palmitoyl Oleoyl 16:0-18:2 Palmitoyl Linoleoyl16:0-20:4 Palmitoyl Arachidonoyl 16:0-22:6 Palmitoyl Docosahexaenoyl18:0-14:0 Stearoyl Myristoyl 18:0-16:0 Stearoyl Palmitoyl 18:0-18:1Stearoyl Oleoyl 18:0-18:2 Stearoyl Linoleoyl 18:0-20:4 StearoylArachidonoyl 18:0-22:6 Stearoyl Docosahexaenoyl 18:1-14:0 OleoylMyristoyl 18:1-16:0 Oleoyl Palmitoyl 18:1-18:0 Oleoyl Stearoyl 14:1-14:1Myristoleoyl Myristoleoyl 14:1-14:1 Myristelaidoyl Myristelaidoyl16:1-16:1 Palmitoleoyl Palmitoleoyl 16:1-16:1 PalmitelaidoylPalmitelaidoyl 18:1-18:1 Petroselinoyl Petroselinoyl 18:1-18:1 OleoylOleoyl 18:1-18:1 Elaidoyl Elaidoyl 18:2-18:2 Linoleoyl Linoleoyl18:3-18:3 Linolenoyl Linolenoyl 20:1-20:1 Eicosenoyl Eicosenoyl20:4-20:4 Arachidonoyl Arachidonoyl 22:1-22:1 Erucoyl Erucoyl 22:6-22:6DHA DHA 24:1-24:1 Nervonoyl Nervonoyl

Linking group: A chemical arm between two compounds, for instance acompound and a label (e.g., an analyte and a label). To accomplish therequisite chemical structure, each of the reactants must contain areactive group. Representative combinations of such groups are aminowith carboxyl to form amide linkages; carboxy with hydroxy to form esterlinkages or amino with alkyl halides to form alkylamino linkages; thiolswith thiols to form disulfides; or thiols with maleimides oralkylhalides to form thioethers. Hydroxyl, carboxyl, amino and otherfunctionalities, where not present in the native compound, may beintroduced by known methods.

Likewise, a wide variety of linking groups may be employed. Thestructure of the linkage should be a stable covalent linkage formed toattach two compounds to each other (e.g., the label to the analyte). Insome cases the linking group may be designed to be either hydrophilic orhydrophobic in order to enhance the desired binding characteristics, forinstance of the modified ligand and its cognate receptor. The covalentlinkages should be stable relative to the solution conditions to whichlinked compounds are subjected. Examples of linking groups will be from1-20 carbons and 0-10 heteroatoms (NH, O, S) and may be branched orstraight chain. Without limiting the foregoing, it should be obviousthat only combinations of atoms that are chemically compatible comprisethe linking group. For example, amide, ester, thioether, thiol ester,keto, hydroxyl, carboxyl, ether groups in combinations withcarbon-carbon bonds are particular examples of chemically compatiblelinking groups.

Operable or contiguous contact: Two solid components are in operablecontact when they are in contact, either directly or indirectly, in sucha manner that an aqueous liquid can flow from one of the two componentsto the other substantially uninterruptedly, by capillarity or otherwise.Direct or contiguous contact means that the two elements are in physicalcontact, such as edge-to-edge or front-to-back. When two components arein direct contact, they may overlap with an overlap of about 0.5 toabout 3 mm. However, the components can be placed with abutting edges.“Indirect contact” means that the two elements are not in physicalcontact, but are bridged by one or more conductors. Operable contact canalso be referred to as “fluid transmitting” or “fluid continuous”contact.

Oxidized cardiolipin (or modified cardiolipin): Cardiolipin that hasbeen chemically modified as described herein. For example, oxidizedcardiolipin is a mixed population of chemically related molecules whereat least one of the four fatty acid chains in non-oxidized cardiolipinhave been oxidatively cleaved to produce a terminal carboxyl group. Insome embodiments, oxidized cardiolipin has a chemical structure shown inFIG. 3.

Reducing agent: Any reducing agent that reduces a β-ketone groupresulting from cardiolipin oxidation to the corresponding β-hydroxylwithout effecting substantial degradation of other oxidized cardiolipinfunctional groups, such as the fatty acid ester groups and/or thephosphate esters. A person of ordinary skill in the art can selectsuitable reducing agents from, for example, those taught by Larock,Comprehensive Organic Transformations, 2nd Edition, New York: John Wiley& Sons, 1999. Particular examples of reducing agents include sodiumbisulfite, dimethyl sulfide, sodium cyanoborohydride (NaBH₃CN), sodiumborohydride (NaBH₄), sodium triacetoxyborohydride (NaBH(OAc)₃),morpholine borane, potassium triisopropoxyborohydride, t-butyl amineborane, dimethylamine borane, pyridine borane, triethylamine borane,trimethylamine borane.

Sample application area: An area where a fluid sample is introduced to aimmunochromatographic test strip, such as an immunochromatographic teststrip present in a lateral flow device. In one example, the sample maybe introduced to the sample application area by external application, aswith a dropper or other applicator. In another example, the sampleapplication area may be directly immersed in the sample, such as when atest strip is dipped into a container holding a sample. In yet anotherexample, the sample may be poured or expressed onto the sampleapplication area.

Solid support (or substrate): Any material which is insoluble, or can bemade insoluble by a subsequent reaction. Numerous and varied solidsupports are known to those in the art and include, without limitation,nitrocellulose, the walls of wells of a reaction tray, multi-wellplates, test tubes, polystyrene beads, magnetic beads, membranes,microparticles (such as latex particles), and sheep (or other animal)red blood cells. Any suitable porous material with sufficient porosityto allow access by detector reagents and a suitable surface affinity toimmobilize capture reagents (e.g., oxidized cardiolipin or oxidizedcardiolipin-attachment molecule complexes) is contemplated by this term.For example, the porous structure of nitrocellulose has excellentabsorption and adsorption qualities for a wide variety of reagents, forinstance, capture reagents. Nylon possesses similar characteristics andis also suitable. Microporous structures are useful, as are materialswith gel structure in the hydrated state.

Further examples of useful solid supports include: natural polymericcarbohydrates and their synthetically modified, cross-linked orsubstituted derivatives, such as agar, agarose, cross-linked alginicacid, substituted and cross-linked guar gums, cellulose esters,especially with nitric acid and carboxylic acids, mixed celluloseesters, and cellulose ethers; natural polymers containing nitrogen, suchas proteins and derivatives, including cross-linked or modifiedgelatins; natural hydrocarbon polymers, such as latex and rubber;synthetic polymers which may be prepared with suitably porousstructures, such as vinyl polymers, including polyethylene,polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and itspartially hydrolyzed derivatives, polyacrylamides, polymethacrylates,copolymers and terpolymers of the above polycondensates, such aspolyesters, polyamides, and other polymers, such as polyurethanes orpolyepoxides; porous inorganic materials such as sulfates or carbonatesof alkaline earth metals and magnesium, including barium sulfate,calcium sulfate, calcium carbonate, silicates of alkali and alkalineearth metals, aluminum and magnesium; and aluminum or silicon oxides orhydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, orglass (these materials may be used as filters with the above polymericmaterials); and mixtures or copolymers of the above classes, such asgraft copolymers obtained by initializing polymerization of syntheticpolymers on a pre-existing natural polymer.

It is contemplated that porous solid supports, such as nitrocellulose,described hereinabove are preferably in the form of sheets or strips.The thickness of such sheets or strips may vary within wide limits, forexample, from about 0.01 to 0.5 mm, from about 0.02 to 0.45 mm, fromabout 0.05 to 0.3 mm, from about 0.075 to 0.25 mm, from about 0.1 to 0.2mm, or from about 0.11 to 0.15 mm. The pore size of such sheets orstrips may similarly vary within wide limits, for example from about0.025 to 15 microns, or more specifically from about 0.1 to 3 microns;however, pore size is not intended to be a limiting factor in selectionof the solid support. The flow rate of a solid support, whereapplicable, can also vary within wide limits, for example from about12.5 to 90 sec/cm (i.e., 50 to 300 sec/4 cm), about 22.5 to 62.5 sec/cm(i.e., 90 to 250 sec/4 cm), about 25 to 62.5 sec/cm (i.e., 100 to 250sec/4 cm), about 37.5 to 62.5 sec/cm (i.e., 150 to 250 sec/4 cm), orabout 50 to 62.5 sec/cm (i.e., 200 to 250 sec/4 cm). In specificembodiments of devices described herein, the flow rate is about 62.5sec/cm (i.e., 250 sec/4 cm). In other specific embodiments of devicesdescribed herein, the flow rate is about 37.5 sec/cm (i.e., 150 sec/4cm).

The surface of a solid support may be activated by chemical processesthat cause covalent linkage of an agent (e.g., a capture reagent) to thesupport. However, any other suitable method may be used for immobilizingan agent (e.g., a capture reagent) to a solid support including, withoutlimitation, ionic interactions, hydrophobic interactions, covalentinteractions and the like. The particular forces that result inimmobilization of an agent on a solid phase are not important for themethods and devices described herein.

A solid phase can be chosen for its intrinsic ability to attract andimmobilize an agent, such as a capture reagent. Alternatively, the solidphase can possess a factor that has the ability to attract andimmobilize an agent, such as a capture reagent. The factor can include acharged substance that is oppositely charged with respect to, forexample, the capture reagent itself or to a charged substance conjugatedto the capture reagent. In another embodiment, a specific binding membermay be immobilized upon the solid phase to immobilize its bindingpartner (e.g., a capture reagent). In this example, therefore, thespecific binding member enables the indirect binding of the capturereagent to a solid phase material.

Except as otherwise physically constrained, a solid support may be usedin any suitable shapes, such as films, sheets, strips, or plates, or itmay be coated onto or bonded or laminated to appropriate inert carriers,such as paper, glass, plastic films, or fabrics.

A “lateral flow substrate” is any solid support or substrate that isuseful in a lateral flow device.

Specific binding partner (or binding partner): A member of a pair ofmolecules that interact by means of specific, noncovalent interactionsthat depend on the three-dimensional structures of the moleculesinvolved. Typical pairs of specific binding partners includeantigen/antibody, hapten/antibody, hormone/receptor, nucleic acidstrand/complementary nucleic acid strand, substrate/enzyme,inhibitor/enzyme, carbohydrate/lectin, biotin/(strept)avidin, andvirus/cellular receptor.

The phrase “specifically binds to an analyte” or “specificallyimmunoreactive with,” when referring to an antibody, refers to a bindingreaction which is determinative of the presence of the analyte in thepresence of a heterogeneous population of molecules such as proteins andother biologic molecules. Thus, under designated immunoassay conditions,the specified antibodies bind to a particular analyte and do not bind ina significant amount to other analytes present in the sample. A varietyof immunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular analyte. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow and Lane,Antibodies, A Laboratory Manual, CSHP, New York (1988), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity.

Subject: Living multi-cellular organisms, including vertebrateorganisms, a category that includes both human and non-human mammals.

Treponemal antigen: An antigen containing at least one antigenicdeterminant that specifically binds anti-T. pallidum antibodies.Numerous treponemal antigens have been described in the art; see, forexample, U.S. Pat. Nos. 6,479,248; 6,248,331; 5,681,934; 5,578,456;4,868,118; and 4,740,467, each of which is incorporated herein byreference. For instance, polypeptides of at least the following apparentmolecular weights have been described as T. pallidum antigens: 16-20kDa, 18 kDa, 18-23 kDa, 25 kDa, 35 kDa, 37 kDa, 37-46 kDa, 38 kDa, 39kDa, 41 kDa, 43 kDa, 44 kDa, 46 kDa, 47 kDa, 58 kDa, 150 kDa; and 180kDa (for more particular detail, see U.S. Pat. No. 4,846,118).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III. Cardiolipin

Cardiolipin is diphosphatidyl glycerol (specifically,1,3-diphosphatidylglycerol), which, as shown below, has a backboneconsisting of three molecules of glycerol joined by two phosphodiesterbridges:

The four hydroxyl groups of cardiolipin's external glycerol moieties areeach esterified with a saturated or unsaturated fatty acid chain(typically from 14 to 18 carbons in length). As used herein, the term“cardiolipin” contemplates 1,3-diphosphatidylglycerol having anydistribution of fatty acid side chains; provided that at least one fattyacid side chain has at least one C═C double bond. Thus, the four fattyacid side chains of cardiolipin can independently vary in length (e.g.,from about 14 to about 25 carbons, from about 14 to about 22 carbons,from about 14 to about 20 carbons, from about 14 to about 18 carbons, orfrom about 14 to about 16 carbons) and/or degree of saturation (e.g.,from completely saturated to having about 6 double bonds, fromcompletely saturated to having about 4 double bonds, or from completelysaturated to having about 2 double bonds). Exemplary fatty acid sidechains of cardiolipin independently include myristoyl (14:0); palmitoyl(16:0); stearoyl (18:0); oleoyl (18:1); myristoleoyl (14:1);palmitoleoyl (16:1); petroselinoyl (18:1); linoleoyl (18:2); linolenoyl(18:3); eicosenoyl (20:1); arachidonoyl (20:4); erucoyl (22:1); DHA(22:6); or nervonoyl (24:1).

In some embodiments, cardiolipin is a naturally occurring form.Naturally occurring cardiolipin is commercially available from a numberof sources, for example, Sigma Aldrich, Avanti Polar Lipids (Alabaster,Ala.), and Lee Laboratories (Grayson, Ga.). It also can be derived, forexample, by solvent extraction of beef heart muscle tissue, by aprecipitation method, or by high pressure column chromatography. Thefatty acid composition of naturally occurring cardiolipin is generallydistributed according to a wide variety of natural occurring fatty acidssuch as palmitoyl (16:0); stearoyl (18:0); oleoyl (18:1); and linoleoyl(18:2). The most abundant fatty acid molecular species in naturallyoccurring forms of cardiolipin are linoleic acid at 90%, followed byoleic acid at 5%, and palmitric acid at 1%.

In other embodiments, cardiolipin is a non-naturally occurring form(also referred to as “synthetic cardiolipin”). Non-limiting examples ofsynthetic cardiolipin include, for example, tetraoleoyl cardiolipin,bis(dipalmitoyl D,L-α-glycerylphosphoryl)-1,3 glycerol benzyl etherdisodium salt, his (dipalmitoyl D,L-α-glycerylphosphoryl)-1,5pentanediol disodium salt, bis(dipalmitoyl D,L-α-glycerylphosphoryl)-1,3propanediol disodium sal, bis(dipalmitoyl D,L-α-glycerylphosphoryl)-1,4butanediol disodium salt, bis (dipalmitoyl D,L-α-glycerylphosphoryl)-1,2ethanediol disodium salt, bis(dipalmitoylD,L-α-glycerylphosphoryl)-methanediol disodium salt, bis(dipalmitoylD,L-α-glycerylphosphoryl)-1,3 glycerol disodium salt,bis(benzylphosphoryl)-1,3-propanediol disodium salt, orD,L-α-dipalmitoyl bis-phosphatidic acid.

The antigenic epitope of cardiolipin is believed to involve the twophosphate groups and the β-hydroxyl group of the central glycerolmoiety. This antigenic epitope is present in both naturally occurringand synthetic cardiolipins. Either or both naturally occurringcardiolipins or synthetic cardiolipins may be oxidized as describedherein as long as anti-lipoidal antibodies will specifically bind to theoxidized form.

IV. Oxidation of Cardiolipin

A. Generally

One of ordinary skill in the art will appreciate that oxidative cleavageof the fatty acid side chains of cardiolipin will occur in the presenceof oxidizing agents, such as NaIO₄ and KMnO₄, or NaIO₄ and rutheniumtetroxide, or HIO₄ and KMnO₄ (see, e.g., March, Advanced OrganicChemistry: Reactions, Mechanisms and Structure, Second Edition, NewYork: McGraw Hill Book Company, 1977, page 1095). That is, the doublebonds present in the fatty acid side chains would be oxidized ultimatelyto carboxyl groups, thereby releasing an alkyl carboxylate, such asmalonic acid and/or caproic acid (as shown in FIG. 3). In a completelyoxidized cardiolipin molecule, all four fatty acid side chains will becleaved at the first (most proximal) double bond position(s) to producefatty acid chains each with a terminal carboxyl group (as shown in FIG.3). Because naturally occurring cardiolipin may be heterogeneous withregard to its fatty acid side chain composition, a completely oxidizedcardiolipin preparation will be a mixture of oxidized species reflectingthe fatty acid side chain heterogeneity in the non-oxidized molecule.Additional heterogeneity may arise if cardiolipin oxidation isincomplete. In that situation, a mixture of oxidized cardiolipin specieswhere 1, 2 or 3 of the fatty acid side chains are oxidized to carboxylicacid(s) will be produced (see, e.g., FIG. 3 for an example of anoxidized cardiolipin molecule with one terminal carboxyl groupillustrative of incomplete oxidation, and another example of an oxidizedcardiolipin which has undergone complete oxidation). Any or all of theoxidized cardiolipin species described in this paragraph arecontemplated herein as being useful in the described compositions,methods, and devices.

B. Oxidation of Cardiolipin by NaIO₄ and KMnO₄

One method useful for the oxidation of cardiolipin employs periodate(IO₄ ⁻) and/or permanganate (MnO₄ ⁻) as the oxidizing agents. Salts ofthese oxidizing agents include any counterion that provides anelectrically neutral compound. The periodate is typically in the form ofa periodate salt, such as NaIO₄ or HIO₄ (periodic acid), and thepermanganate is typically in the form of a salt (such as a sodium orpotassium salt), for example KMnO₄. In one specific embodiment, NaIO₄and KMnO₄ are used as oxidizing agents. To practice this oxidationmethod, cardiolipin obtained from any source may be added to any solventin which the cardiolipin will substantially dissolve. In view of thehydrophobic nature of cardiolipin, the use of a polar organic solvent,such as t-butanol, ethanol, methanol, propanol, acetone,dimethylformamide, or diethyl ether is preferable. Other non-polarorganic solvents may be used to suspend cardiolipin, such as chloroform;however, these non-polar organic solvents are less useful because othercomponents of the oxidation reaction (as described below) are lesssoluble in these solvents than, for example, a polar organic solvent oran aqueous solvent.

The concentration of the cardiolipin solution may be any usefulconcentration that, in the upper range, still permits cardiolipin todissolve in the chosen solvent. One of skill in the art can easilydetermine the saturation point for cardiolipin in any particularsolvent. In examples where t-butanol is the chosen solvent, cardiolipinconcentrations may be in the range of about 10 mg/ml to 250 mg/ml, suchas about 10 mg/ml, about 25 mg/ml, about 50 mg/ml, about 75 mg/ml, about100 mg/ml, about 125 mg/ml, about 150 mg/ml, about 200 mg/ml and about250 mg/ml. If particulates are present after dissolving cardiolipin inthe chosen solvent, the solution may, optionally, be clarified by anytechnique known in the art, such as centrifugation or filtration.

In some embodiments, oxidation of cardiolipin may take place in theabsence of oxygen, for example in an argon, helium, or nitrogenatmosphere. A cardiolipin oxidation reaction will occur in anoxygen-containing atmosphere, such as air, or other atmosphere; however,oxidizing molecules contained therein, such as carbonates, may decreasethe efficiency of the reaction. Nonetheless, all atmospheres in whichthe cardiolipin oxidation reaction will occur, regardless of reactionefficiency, are contemplated by this disclosure.

Amounts of oxidizing agents such as NaIO₄ and KMnO₄, sufficient tooxidatively cleave at least one cardiolipin fatty acid side chain arethen added to the suspended cardiolipin with constant stirring. In someexamples, the periodate and permanganate (such as NaIO₄ and KMnO₄) areused in combination to oxidize cardiolipin. In certain of thoseexamples, the periodate (such as NaIO₄) is added to the cardiolipinsolution prior to the addition of the permanganate (such as KMnO₄). Theoxidizing agents may be dissolved in any solvent in which they aresoluble and that will mix with the cardiolipin solution. For example,NaIO₄ and KMnO₄ may be dissolved in water.

Where NaIO₄ is used in a method to oxidize cardiolipin, the amount ofNaIO₄ used in the reaction is not critical. Minimally, the amount issuch that the reaction takes place in a reasonable time under theparticular circumstances. At the other end of the spectrum, aconsiderable molar excess of NaIO₄ may be used in the oxidationreaction. For example, the molar ratio of periodate (such as NaIO₄) tocardiolipin can be about 0.1:1 to about 100:1, about 0.5:1 to about50:1, about 1:1 to about 25:1, about 2:1 to about 15:1, about 2.5:1 toabout 10:1, about 3:1 to about 7.5:1, or about 4:1 to about 5:1. Inspecific embodiments, the molar ratio of sodium m-periodate tocardiolipin is about 4:1 to 5:1, or more specifically about 4.2:1.

Where permanganate (such as KMnO₄) is used in a method to oxidizecardiolipin, the amount of permanganate used in the reaction is notcritical. Minimally, the amount is such that the reaction takes place ina reasonable time under the particular circumstances. At the other endof the spectrum, a considerable molar excess of permanganate may be usedin the oxidation reaction. For example, the molar ratio of permanganate(such as KMnO₄) to cardiolipin can be about 0.01:1 to about 100:1, about0.02:1 to about 50:1, about 0.1:1 to about 25:1, about 0.25:1 to about15:1, about 0.3:1 to about 10:1, about 0.4:1 to about 7.5:1, about 0.5:1to about 5:1, about 0.6:1 to about 2:1, or about 0.7:1 to about 1:1. Inspecific embodiments, the molar ratio of the permanganate (such asKMnO₄) to cardiolipin is about 0.5:1 to 1:1, or more specifically about0.75:1.

The oxidation reaction mixture may take place at any temperature thatdoes not stop the reaction from occurring. Similarly, the reaction mayproceed for any amount of time that is sufficient to cause oxidativecleavage of at least one fatty acid side chain of cardiolipin. As one ofskill in the art will appreciate, reaction time will depend upon severalvariables, including reaction temperature, type of solvent, and reactantand product concentrations, each of which variables may be easilyoptimized with routine experimentation. In one example, the oxidationreaction occurs at room temperature and proceeds for at least 24-48hours.

The cardiolipin oxidation reaction may be stopped with any reducingagent capable of neutralizing the oxidizing agent(s) present in thereaction and capable of reducing any ketone formed at the β-carbon ofthe central glycerol moeity to the corresponding β-hydroxyl group. Areducing agent, such as a bisulfite salt (such as, sodium bisulfite),dimethyl sulfide, sodium cyanoborohydride (NaBH₃CN), sodium borohydride(NaBH₄), sodium triacetoxyborohydride (NaBH(OAc)₃), morpholine borane,potassium triisopropoxyborohydride, t-butyl amine borane, dimethylamineborane, pyridine borane, triethylamine borane, or trimethylamine borane,is useful for this purpose. In some specific examples, the reducingagent is a bisulfite salt, and in more particular examples, the reducingagent is sodium bisulfite.

The amount of reducing agent added to the reaction is not critical aslong as the oxidizing agents are quenched. If immunogenicity of oxidizedcardiolipin is adversely affected by oxidation to a ketone of theβ-hydroxyl groups of cardiolipin central glycerol moieties, then anamount of reducing agent is also an amount that will restore β-hydroxylgroups for sufficient immunogenicity of the oxidized cardiolipin. Forexample, an amount of sodium bisulfite may be added to a cardiolipinoxidation reaction containing NaIO₄ and KMnO₄ oxidizing agentssufficient to cause the reaction mixture to turn substantiallycolorless.

If present, aqueous and organic phases in a cardiolipin oxidationreaction may be separated by any method known in the art, for examplecentrifugation. In one embodiment, a t-butanol phase containingprimarily oxidized cardiolipin and a water phase containing primarilyalkyl carboxylates may be separated by centrifugation. Oxidizedcardiolipin is sufficiently hydrophobic that it may be expected toseparate into an organic phase. However, if necessary, an ordinarilyskilled artisan may determine which of an aqueous or organic phase (orboth) contains oxidized cardiolipin by methods known in the art, such asTLC, HPLC, NMR or gas chromatography.

Optionally, oxidized cardiolipin in solution may be dialyzed into auseful buffer, such as 10 mM phosphate buffer, pH 8.0, and lyophilizedto dryness using methods well known in the art.

C. Antigenicity of Oxidized Cardiolipin

Oxidation has been shown to alter the antigenic properties of somephospholipids (see, e.g., U.S. Pat. No. 6,177,282). It is believed thatanti-lipoidal antibodies of syphilitic patients bind the two phosphategroups and the β-hydroxyl group of the central glycerol moiety (see,e.g., Castro et al., Clin. Diagn. Lab. Immunol., 7(4):658-661, 2000).Thus, it is beneficial to maintain the configuration of the centralglycerol moiety in order to retain cardiolipin antigenicity, forexample, to detect anti-lipoidal antibodies in syphilitic sera. Theantigenicity of oxidized cardiolipin may be tested by any of a number ofwidely used techniques (including, e.g., ELISA, dot blot, enzymeimmunoassay, fluorescence immunoassay) and/or as described herein (see,e.g., Example 4). As shown in Example 4 (below), methods of oxidizingcardiolipin described herein maintain cardiolipin antigenicity.

D. Cardiolipin/Lecithin Mixtures

Mixtures of cardiolipin and lecithin may be oxidized by the same methodsdescribed above for cardiolipin alone; the only difference being thatthe starting material is a mixture of cardiolipin and lecithin. In theinitial oxidation reaction, cardiolipin and lecithin are mixed togetherin a weight ratio (cardiolipin:lecithin) between about 20:1 to about1:1, or between about 1:1 to about 1:10, or between about 10:1 to about1:10, or between about 5:1 to about 1:5, or between about 1:1 to about1:5. In particular examples, the weight ratio of cardiolipin:lecithinincludes about 20:1, about 10:1, about 5:1, about 2:1, about 1:1, about1:2, about 1:3, about 1:5, or about 1:10.

V. Oxidized Cardiolipin-Attachment Molecule Complex

Oxidized cardiolipin is a relatively small molecule. Thus, it isbeneficial to attach oxidized cardiolipin to a larger molecule (such asa polypeptide) to facilitate the attachment of cardiolipin to a solidsurface for use in the methods and devices described herein. In certainexamples, the larger molecule is more readily attached to a substrate,such as a bibulous substrate of the type that is used in lateral flow orflow-through technology. The derivatized oxidized cardiolipin is morereadily adsorbed and localized to a porous substrate (such as anitrocellulose porous substrate) than the very small cardiolipin oroxidized cardiolipin molecule itself. Derivatizing the oxidizedcardiolipin by attaching a polypeptide moiety to it greatly enhances itsversatility and usefulness in solid-surface-based immunoassays, such asELISAs, lateral flow diagnostic strips and/or devices, and/orflow-through devices).

Suitable attachment molecules include proteins, polypeptides or peptidessuch as albumin, hemocyanin, thyroglobulin and derivatives thereof,particularly bovine serum albumin (BSA) and keyhole limpet hemocyanin(KLH), avidin, streptavidin, and biotin. Other polypeptide-derived ornon-polypeptide derived substances are known to those skilled in theart.

Attachment molecules often contain a reactive group to facilitatecovalent conjugation to the oxidized cardiolipin. The amine group ofamino acids can be used in this manner. Attachment molecules lackingsuch groups can often be reacted with an appropriate chemical to producereactive groups. Illustrative chemicals that can be used to produceuseful reactive groups in an attachment molecule include, withoutlimitation 1-ethyl-3-(3-dimethylamino propyl)carbodiimide (EDC), andN-hydroxysulfosuccinimide (NHS).

The provision of oxidized cardiolipin herein provides at least onereactive carboxyl group, which is not otherwise available in naturallyoccurring or synthetic cardiolipins. Thus, oxidized cardiolipin may belinked via an oxidation-created reactive group to an attachment moleculeby any means known in the art and/or as described herein. Such linkagemay be either with or without an additional linking group. Manydifferent methods may be used to produce a linkage between oxidizedcardiolipin and an attachment molecule.

One example method that may be used to link oxidized cardiolipin to anamine-containing attachment molecule (e.g., a protein, such as BSA,synthetic protein MAPS, IgY, strepavidin, avidin or KLH) is shownschematically in FIG. 4. The reaction in FIG. 4 illustrates that acarboxyl group created by oxidative cleavage of a cardiolipin fatty acidside chain, as described herein, may be modified to an amine-reactiveNHS ester using EDC in combination with NHS (or Sulfo-NHS, as shown inFIG. 4). The oxidized cardiolipin NHS ester will then react with aminegroups present in an attachment molecule, such as BSA as shown in FIG.4, to form an amide linkage between oxidized cardiolipin and theattachment molecule.

Some embodiments of the methods and devices herein make use of oxidizedcardiolipin or oxidized cardiolipin-attachment molecule compleximmobilized on solid phases. Any conventional method of immobilizing asubstance on a solid surface is contemplated in this disclosure.

Suitable methods for immobilizing oxidized cardiolipin or oxidizedcardiolipin-attachment molecule complex on solid phases include ionic,hydrophobic, covalent interactions and the like. The solid phase (see,e.g., Section II) can be chosen for its intrinsic ability to attract andimmobilize oxidized cardiolipin or oxidized cardiolipin-attachmentmolecule complex. Alternatively, the solid phase can possess a factorthat has the ability to attract and immobilize oxidized cardiolipin oroxidized cardiolipin-attachment molecule complex. The factor caninclude, for example, a charged substance that is oppositely chargedwith respect to oxidized cardiolipin or oxidized cardiolipin-attachmentmolecule complex, or, for example, a charged substance that isoppositely charged with respect to a charged substance conjugated tooxidized cardiolipin or oxidized cardiolipin-attachment moleculecomplex. As yet another alternative, the factor can be any specificbinding partner, for example, avidin or streptavidin, which isimmobilized upon the solid phase and which has the ability to immobilizeoxidized cardiolipin or oxidized cardiolipin-attachment moleculecomplex, for example, biotinylated oxidized cardiolipin, through aspecific binding reaction.

In some embodiments of methods and devices herein, oxidized cardiolipinor oxidized cardiolipin-attachment molecule complex may serve as adetector reagent, and thus will be conjugated to a label. Any moleculeor composition that is detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means mayserve as a label. Examples of labels, including enzymes, colloidal goldparticles, colored latex particles, have been disclosed (U.S. Pat. Nos.4,275,149; 4,313,734; 4,373,932; and 4,954,452, each incorporated byreference herein). Additional examples of useful labels include, withoutlimitation, co-factors, ligands, chemiluminescent or fluorescent agents,protein-adsorbed silver particles, protein-adsorbed iron particles,protein-adsorbed copper particles, protein-adsorbed selenium particles,protein-adsorbed sulfur particles, protein-adsorbed tellurium particles,protein-adsorbed carbon particles, and protein-coupled dye sacs.

The attachment of a compound to a label can be through covalent bonds,adsorption processes, hydrophobic and/or electrostatic bonds, as inchelates and the like, or combinations of these bonds and interactions,and/or may involve a linking group. Methods for labeling and guidance inthe choice of labels appropriate for various purposes are discussed, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual,CSHL, New York, 1989 and Ausubel et al., Current Protocols in MolecularBiology, Greene Publ. Assoc. and Wiley-Intersciences, 1998.

In some embodiments of methods and devices, colloidal gold conjugates ofoxidized cardiolipin or colloidal gold conjugates of oxidizedcardiolipin-attachment molecule complexes are envisaged.

VI. Immunoassay Devices

The discovery herein of a method to oxidize cardiolipin and preparecardiolipin-attachment molecule conjugates, which can be immobilized ona solid support (such as a microporous membrane, like nitrocellulose,nylon or PVDF) enables solid-surface-based immunoassays (such as, EIA,ELISA, flow-through devices, dipsticks, and lateral flow devices) forthe detection of cardiolipin-binding analytes (such as anti-lipoidalantibodies in biological samples from T. pallidum-infected subjects). Insome examples, a disclosed immunoassay permits detection of the presence(or absence) of anti-lipoidal antibodies in a biological sample fordiagnosis of syphilis.

A. Representative Immunoassay Device Formats and Related Information

Immunoassay devices permit the performance of relatively inexpensive,disposable, membrane-based assays for the visual identification of thepresence (or absence) of an analyte in a liquid sample. Such devices areusually formatted as freestanding dipsticks (e.g., test strips) or asdevices having some sort of housing. Typically, an immunoassay devicecan be used with as little as about 200 μl of liquid sample, anddetection of an analyte in the sample can (but need not) be completewithin 2-5 minutes. In clinical assays, the sample may be urine, blood,serum, saliva, or other body fluids. In nonclinical tests, the samplemay be a small volume of solution prepared from soil, dust, plants, orfood, and similarly applied directly to the membrane test strip. In mostinstances, no ancillary instrumentation is required to perform suchtests, and such devices easily can be used in clinics, laboratories,field locations, and the home even by inexperienced persons.

Immunoassay devices have been developed for the routine identificationor monitoring of physiological and pathological conditions (e.g.,infectious diseases, pregnancy, cancer, endocrine disorders) usingdifferent biological samples (e.g., urine, serum, plasma, blood,saliva), and for analysis of environmental samples (e.g., natural fluidsand industrial plant effluents) for instance for contamination. Many ofthese tests are based on the highly specific interactions betweenspecific binding pairs. Examples of such binding pairs includeantigen/antibody, hapten/antibody, lectin/carbohydrate,apoprotein/cofactor and biotin/(strept)avidin. Furthermore, many ofthese tests involve devices (e.g., solid phase, lateral flow teststrips, flow-through tests) with one or more of the members of a bindingpair attached to a mobile or immobile solid phase material such as latexbeads, glass fibers, glass beads, cellulose strips or nitrocellulosemembranes (U.S. Pat. Nos. 4,703,017; 4,743,560; 5,073,484).

One principle category of immunochromatographic assay is the “sandwich”assay. In general, sandwich immunochromatographic procedures call formixing the sample that may contain the analyte to be assayed, forexample, anti-lipoidal antibody, with an antigen recognized by theanalyte, for example, oxidized cardiolipin. The antigen, i.e., detectorreagent, is mobile and typically is linked to a label or anothersignaling reagent, such as dyed latex, a colloidal metal sol, or aradioisotope. This mixture is then applied to a chromatographic mediumcontaining a band or zone of immobilized antigens recognized by theanalyte antibody of interest. The chromatographic medium often is in theform of a strip that resembles a dipstick. When the complex of themolecule to be assayed and the detector reagent reaches the zone of theimmobilized antigens on the chromatographic medium, binding occurs andthe detector reagent complex is localized at the zone. This indicatesthe presence of the molecule to be assayed. This technique can be usedto obtain quantitative or semi-quantitative results.

Alternatively, a sandwich immunoassay may call for mixing a sample,which may contain the analyte of interest, for example, anti-lipoidalantibody, with an antibody that recognizes the analyte, for example,protein A or a goat anti-human secondary antibody. The secondaryantibody in this mixture will be mobile, labeled (with, e.g., an enzyme,colloidal gold, or other) and serve as a detector reagent. As describedin the previous example of a sandwich immunoassay, the assayed analyte(if present) may be detected when this mixture is applied to achromatographic medium containing a band or zone of immobilized antigensrecognized by the assayed.

Examples of sandwich immunoassays performed on test strips are describedin U.S. Pat. Nos. 4,168,146 and 4,366,241, each of which is incorporatedherein by reference.

Solid phase immunoassay devices provide sensitive detection of analytesin biological fluid samples. Solid phase immunoassay devices incorporatea solid support to which one member of a ligand-receptor pair, usuallyan antibody, antigen, or hapten, is bound. Common early forms of solidsupports were plates, tubes, or beads of polystyrene, which were knownfrom the fields of radioimmunoassay and enzyme immunoassay. Morerecently, a number of porous materials such as nylon, nitrocellulose,cellulose acetate, glass fibers, and other porous polymers have beenemployed as solid supports. In other common forms of membrane-basedimmunoassays, as typified by some home pregnancy and ovulation detectionkits, a test strip (or dipstick) is “dipped” into a sample suspected ofcontaining the subject analyte. Enzyme-labeled detector reagent is thenadded, either simultaneously or after an incubation period. The devicenext is washed and then inserted into a second solution containing asubstrate for the enzyme. The enzyme label, if present, interacts withthe substrate, causing the formation of colored products, which eitherdeposit as a precipitate onto the solid phase or produce a visible colorchange in the substrate solution. EP-A 0 125 118 describes such asandwich type dipstick immunoassay. EP-A 0 282 192 describes a dipstickdevice for use in competition type assays.

Flow-through type immunoassay devices were designed, in part, to obviatethe need for incubation and washing steps associated with dipstickassays. Flow-through immunoassay devices involve a capture reagent (suchas an oxidized cardiolipin-attachment molecule complex) bound to aporous membrane or filter to which a liquid sample is added. As theliquid flows through the membrane, target analyte (such as,anti-lipoidal antibody) binds to the capture reagent. The addition ofsample is followed by (or made concurrent with) addition of detectorreagent (such as, gold-conjugated cardiolipin, labeled (e.g.,gold-conjugated) Protein A or labeled (e.g., gold-conjugate) anti-humanIgG). Alternatively, the detector reagent may be placed on the membranein a manner that permits the detector to mix with the sample and therebylabel the analyte. The visual detection of detector reagent provides anindication of the presence of target analyte in the sample.Representative flow-through immunoassay devices are described in U.S.Pat. Nos. 4,246,339; 4,277,560; 4,632,901; 4,812,293; 4,920,046; and5,279,935; and U.S. Patent Application Publication Nos. 20030049857 and20040241876.

Migration assay devices usually incorporate within them reagents thathave been attached to colored labels, thereby permitting visibledetection of the assay results without addition of further substances.See, for example, U.S. Pat. No. 4,770,853; PCT Publication No. WO88/08534 and European Patent No. EP-A 0 299 428.

There are a number of commercially available lateral flow type tests andpatents disclosing methods for the detection of large analytes (MWgreater than 1,000 Daltons). U.S. Pat. No. 5,229,073 describes asemiquantitative competitive immunoassay lateral flow method formeasuring plasma lipoprotein levels. This method utilizes a plurality ofcapture zones or lines containing immobilized antibodies to bind boththe labeled and free lipoprotein to give a semi-quantitative result.

U.S. Pat. No. 5,591,645 provides a chromatographic test strip with atleast two portions. The first portion includes a movable tracer and thesecond portion includes an immobilized binder capable of binding to theanalyte. Additional examples of lateral flow tests for large analytesare disclosed in the following patent documents: U.S. Pat. Nos.4,168,146; 4,366,241; 4,855,240; 4,861,711; and 5,120,643; EuropeanPatent No. 0296724; WO 97/06439; and WO 98/36278.

There are also lateral flow type tests for the detection ofsmall-analytes (MW 100-1,000 Daltons). Generally, these small analytetests involve “typical” competitive inhibition to produce negative orindirect reporting results (i.e., reduction of signal with increasinganalyte concentration), as exemplified by U.S. Pat. No. 4,703,017.However, several approaches have been developed for detecting smallanalytes using lateral flow tests that produce positive or directreporting results (i.e., increase in signal with increasing analyteconcentration). These include, for instance, U.S. Pat. Nos. 5,451,504;5,451,507; 5,798,273; and 6,001,658.

U.S. Pat. No. 5,451,504 provides a method with three specific zones(mobilization, trap and detection) each containing a different latexconjugate to yield a positive signal. The mobilization zone containslabeled antibody to bind the analyte in the sample. In the trap zone,unbound, labeled antibody is then trapped by immobilized analyte analog.The detection zone captures the labeled analyte-antibody complex.

U.S. Pat. No. 5,451,507 describes a two-zone, disconnected immunoassaymethod. The first zone has non-diffusively bound reagent that binds witha component, for example, an analyte analog bound to, or capable ofbecoming bound to, a member of a signal producing system. The secondzone binds to the component only when the analyte to be tested ispresent. The distance the component migrates into the second zone isdirectly related to the concentration of analyte.

U.S. Pat. No. 5,798,273 discloses a lateral flow device that includes acapture zone with immobilized analyte analog and one or more read-outzones to bind labeled analyte-analog.

U.S. Pat. No. 6,001,658 discloses a test strip device with a diffusible,labeled binding partner that binds with analyte, an immobilized analyte,and a detection area containing an immobilized antibody.

Devices described herein generally include a strip of absorbent material(such as a microporous membrane), which, in some instances, can be madeof different substances each joined to the other in zones, which may beabutted and/or overlapped. In some examples, the absorbent strip can befixed on a supporting non-interactive material (such as nonwovenpolyester), for example, to provide increased rigidity to the strip.Zones within each strip may differentially contain the specific bindingpartner(s) and/or other reagents required for the detection and/orquantification of the particular analyte being tested for, for example,an anti-lipoidal antibody. Thus these zones can be viewed as functionalsectors or functional regions within the test device.

In general, a fluid sample (or a sample suspended in a fluid) isintroduced to the strip at the proximal end of the strip, for instanceby dipping or spotting. A sample is collected or obtained using methodswell known to those skilled in the art. The sample containing theanti-lipoidal antibodies to be detected may be obtained from anybiological source. Examples of biological sources include blood serum,blood plasma, urine, spinal fluid, saliva, fermentation fluid, lymphfluid, tissue culture fluid and ascites fluid of a human or animal. Thesample may be diluted, purified, concentrated, filtered, dissolved,suspended or otherwise manipulated prior to immunoassay to optimize theimmunoassay results. The fluid migrates distally through all thefunctional regions of the strip. The final distribution of the fluid inthe individual functional regions depends on the adsorptive capacity andthe dimensions of the materials used.

In some embodiments, porous solid supports, such as nitrocellulose,described hereinabove are preferably in the form of sheets or strips.The thickness of such sheets or strips may vary within wide limits, forexample, from about 0.01 to 0.5 mm, from about 0.02 to 0.45 mm, fromabout 0.05 to 0.3 mm, from about 0.075 to 0.25 mm, from about 0.1 to 0.2mm, or from about 0.11 to 0.15 mm. The pore size of such sheets orstrips may similarly vary within wide limits, for example from about0.025 to 15 microns, or more specifically from about 0.1 to 3 microns;however, pore size is not intended to be a limiting factor in selectionof the solid support. The flow rate of a solid support, whereapplicable, can also vary within wide limits, for example from about12.5 to 90 sec/cm (i.e., 50 to 300 sec/4 cm), about 22.5 to 62.5 sec/cm(i.e., 90 to 250 sec/4 cm), about 25 to 62.5 sec/cm (i.e., 100 to 250sec/4 cm), about 37.5 to 62.5 sec/cm (i.e., 150 to 250 sec/4 cm), orabout 50 to 62.5 sec/cm (i.e., 200 to 250 sec/4 cm). In specificembodiments of devices described herein, the flow rate is about 62.5sec/cm (i.e., 250 sec/4 cm). In other specific embodiments of devicesdescribed herein, the flow rate is about 37.5 sec/cm (i.e., 150 sec/4cm).

Another common feature to be considered in the use of immunoassaydevices is a means to detect the formation of a complex between ananalyte (such as an anti-lipoidal antibody) and a capture reagent (suchas oxidized cardiolipin-attachment molecule complexes). A detector (alsoreferred to as detector reagent) serves this purpose. A detector may beintegrated into an immunoassay device (for example included in aconjugate pad, as described below), or may be applied to the device froman external source.

A detector may be a single reagent or a series of reagents thatcollectively serve the detection purpose. In some instances, a detectorreagent is a labeled binding partner specific for the analyte (such as,gold-conjugated Protein A for an antibody analyte, or gold-labeledanti-human Ab(Fc) for a human antibody analyte, or gold-labeled oxidizedcardiolipin for an anti-lipoidal antibody analyte). In other instances,a detector reagent collectively includes an unlabeled first bindingpartner specific for the analyte and a labeled second binding partnerspecific for the first binding partner and so forth. In each instance, adetector reagent specifically detects bound analyte of ananalyte-capture reagent complex and, therefore, a detector reagentpreferably does not substantially bind to or react with the capturereagent or other components localized in the analyte capture area. Suchnon-specific binding or reaction of a detector may provide a falsepositive result. Optionally, a detector reagent can specificallyrecognize a positive control molecule (such as a non-specific human IgGfor a labeled Protein A detector, or a labeled Protein G detector, or alabeled anti-human Ab(Fc)) that is present in a secondary capture area.

B. Flow-Through Device Construction and Design

A flow-through device involves a capture reagent (such as oxidizedcardiolipin-attachment molecule complex) immobilized on a solid support,typically, microtiter plate or a membrane (such as, nitrocellulose,nylon, or PVDF). Characteristics of useful membrane have been previouslydescribed; however, it is useful to note that in a flow-through assaycapillary rise is not a particularly important feature of a membrane asthe sample moves vertically through the membrane rather than across itas in a lateral flow assay. In a simple representative format, themembrane of a flow-through device is placed in functional or physicalcontact with an absorbent layer (see, e.g., description of “absorbentpad” below), which acts as a reservoir to draw a fluid sample throughthe membrane. Optionally, following immobilization of a capture reagent,any remaining protein-binding sites on the membrane can be blocked(either before or concurrent with sample administration) to minimizenonspecific interactions.

In operation of a flow-through device, a fluid sample (such as a bodilyfluid sample) is placed in contact with the membrane. Typically, aflow-through device also includes a sample application area (orreservoir) to receive and temporarily retain a fluid sample of a desiredvolume. The sample passes through the membrane matrix. In this process,an analyte in the sample (such as an anti-lipoidal antibody) canspecifically bind to the immobilized capture reagent (such as oxidizedcardiolipin-attachment molecule complex). Where detection of ananalyte-capture reagent complex is desired, a detector reagent (such aslabeled Protein A, labeled Protein G, labeled anti-human IgG, or labeledcardiolipin) can be added with the sample or a solution containing adetector reagent can be added subsequent to application of the sample.If an analyte is specifically bound by capture reagent, a visualrepresentative attributable to the particular detector reagent can beobserved on the surface of the membrane. Optional wash steps can beadded at any time in the process, for instance, following application ofthe sample, and/or following application of a detector reagent.

C. Lateral Flow Device Construction and Design

Lateral flow devices are commonly known in the art. Briefly, a lateralflow device is an analytical device having as its essence a test strip,through which flows a test sample fluid that is suspected of containingan analyte of interest. The test fluid and any suspended analyte canflow along the strip to a detection zone in which the analyte (ifpresent) interacts with a capture agent and a detection agent toindicate a presence, absence and/or quantity of the analyte.

Numerous lateral flow analytical devices have been disclosed, andinclude those shown in U.S. Pat. Nos. 4,313,734; 4,435,504; 4,775,636;4,703,017; 4,740,468; 4,806,311; 4,806,312; 4,861,711; 4,855,240;4,857,453; 4,943,522; 4,945,042; 4,496,654; 5,001,049; 5,075,078;5,126,241; 5,451,504; 5,424,193; 5,712,172; 6,555,390; and 6,368,876; EP0810436; and WO 92/12428; WO 94/01775; WO 95/16207; and WO 97/06439,each of which is incorporated by reference.

Many lateral flow devices are one-step lateral flow assays in which abiological fluid is placed in a sample area on a bibulous strip (though,non-bibulous materials can be used, and rendered bibulous, e.g., byapplying a surfactant to the material), and allowed to migrate along thestrip until the liquid comes into contact with a specific bindingpartner that interacts with an analyte in the liquid. Once the analyteinteracts with the binding partner, a signal (such as a fluorescent orotherwise visible dye) indicates that the interaction has occurred.Multiple discrete binding partners can be placed on the strip (forexample in parallel lines) to detect multiple analytes in the liquid.The test strips can also incorporate control indicators, which provide asignal that the test has adequately been performed, even if a positivesignal indicating the presence (or absence) of an analyte is not seen onthe strip.

The construction and design of lateral flow devices is very well knownin the art, as described, for example, in Millipore Corporation, A ShortGuide Developing Immunochromatographic Test Strips, 2nd Edition, pp.1-40, 1999, available by request at (800) 645-5476; and Schleicher &Schuell, Easy to Work with BioScience, Products and Protocols 2003, pp.73-98, 2003, 2003, available by request at Schleicher & SchuellBioScience, Inc., 10 Optical Avenue, Keene, N.H. 03431, (603) 352-3810;both of which are incorporated herein by reference.

Lateral flow devices have a wide variety of physical formats that areequally well known in the art. Any physical format that supports and/orhouses the basic components of a lateral flow device in the properfunction relationship is contemplated by this disclosure. FIG. 7 showsseveral examples of lateral flow devices. These examples demonstratesome of the physical embodiments that may be useful in the constructionof a lateral flow device.

The basic components of a particular embodiment of a lateral flow deviceare illustrated in FIG. 8, which shows a particular embodiment in whichan elongated housing 10 contains a bibulous lateral flow strip 12 thatextends substantially the entire length of housing 10. Lateral flowstrip 12 is divided into a proximal sample application pad 14 positionedbelow a sample introduction port 15, an intermediate test resultmembrane 16, and a distal absorbent pad 18. Flow strip 12 is interruptedby a conjugate pad 20 that contains labeled conjugate (such asgold-conjugated Protein A, gold-conjugated Protein G, gold-conjugatedanti-human Ab). A flow path along strip 12 passes from proximal pad 14,through conjugate pad 20, into test result membrane 16, for eventualcollection in absorbent pad 18. Selective binding agents (such as ananchor antibody-lipoidal antigen complex) are positioned on a proximaltest line 22 in test result membrane 16. A control line 24 is providedin test result membrane 16 slightly distal to test line 22.

In operation of the particular embodiment of a lateral flow deviceillustrated in FIG. 8, a fluid sample containing an analyte of interest,such as an anti-lipoidal antibody, is applied to the sample pad 14through the sample introduction port 15. In some examples, the samplemay be applied to the sample introduction port 15 dropwise or, lesspreferably, by dipping the end of the device containing the sampleintroduction port 15 into the sample. In other examples where a sampleis whole blood, an optional developer fluid is added to the blood sampleto cause hemolysis of the red blood cells and, in some cases, to make anappropriate dilution of the whole blood sample. From the sample pad 14,the sample passes, for instance by capillary action, to the conjugatepad 20. In the conjugate pad 20, the analyte of interest may bind (or bebound by) a mobilized or mobilizable detector reagent. For example, ananti-lipoidal antibody analyte may bind to a labeled (e.g.,gold-conjugated) Protein A or gold-conjugated cardiolipin detectorreagent contained in the conjugate pad. The analyte complexed with thedetector reagent may subsequently flow to the test result membrane 16where the complex may further interact with an analyte-specific bindingpartner (such as oxidized cardiolipin-attachment molecule complex),which is immobilized at the proximal test line 22. In some examples, ananti-lipoidal antibody complexed with a detector reagent (such as,gold-conjugated cardiolipin, labeled (e.g., gold-conjugated) Protein A,labeled (e.g., gold-conjugated) Protein G, labeled (e.g.,gold-conjugated) anti-human Ab) may further bind to unlabeled, oxidizedcardiolipin-attachment molecule complexes immobilized at the proximaltest line 22. The formation of the immunocomplex between anti-lipoidalantibody, labeled (e.g., gold-conjugated) detector reagent, andimmobilized oxidized cardiolipin-attachment molecule complex can bedetected by the appearance of a visible line at the proximal test line22, which results from the accumulation of the label (e.g., gold) in thelocalized region of the proximal test line 22. The control line 24 maycontain an immobilized, detector-reagent-specific binding partner, whichcan bind the detector reagent in the presence or absence of the analyte.Such binding at the control line 24 indicates proper performance of thetest, even in the absence of the analyte of interest.

In another embodiment of a lateral flow device, there may be a secondtest line located parallel or perpendicular (or in any other spatialrelationship) to test line 22 in test result membrane 16. The operationof this particular embodiment is similar to that described in theimmediately preceding paragraph with the additional considerations that(i) a second detector reagent specific for a second analyte, such as ananti-T pallidum antibody or T. pallidum organism or antigen, may also becontained in the conjugate pad, and (ii) the second test line willcontain a second specific binding partner having affinity for a secondanalyte in the sample. For example, the second test line may containimmobilized treponemal antigens that will specifically bind anti-T.pallidum antibodies present in the sample or contain immobilized anti-T.pallidum antibodies that will specifically bind T. pallidum antigens ororganisms present in the sample.

Some of the materials that may be useful for the components of a lateralflow device are shown in Table 1. However, one of skill in the art willrecognize that the particular materials used in a particular lateralflow device will depend on a number of variables, including, forexample, the analyte to be detected, the sample volume, the desired flowrate and others, and can routinely select the useful materialsaccordingly.

TABLE 1 Component Useful Material Sample Pad Glass fiber Woven fibersScreen Non-woven fibers Cellulosic filters Paper Conjugate Pad Glassfiber Polyester Paper Surface modified polypropylene MembraneNitrocellulose (including pure nitrocellulose and modifiednitrocellulose) Nitrocellulose direct cast on polyester supportPolyvinylidene fluoride Nylon Absorbent Pad Cellulosic filters Paper

1. Sample Pad

The sample pad (such as sample pad 14 in FIG. 8) is an optionalcomponent of a lateral flow device that initially receives the sample,and may serve to remove particulates from the sample. Among the variousmaterials that may be used to construct a sample pad (see Table 1), acellulose sample pad may be beneficial if a large bed volume (e.g., 250μl/cm²) is a factor in a particular application. Sample pads may betreated with one or more release agents, such as buffers, salts,proteins, detergents, and surfactants. Such release agents may beuseful, for example, to promote resolubilization of conjugate-padconstituents, and to block non-specific binding sites in othercomponents of a lateral flow device, such as a nitrocellulose membrane.Representative release agents include, for example, trehalose or glucose(1%-5%), PVP or PVA (0.5%-2%), Tween 20 or Triton X-100 (0.1%-1%),casein (1%-2%), SDS (0.02%-5%), and PEG (0.02%-5%).

2. Membrane and Application Solution:

The types of membranes useful in a lateral flow device (such asnitrocellulose, nylon and PVDF), and considerations for applying acapture reagent to such membranes have been discussed previously.

3. Conjugate Pad

The conjugate pad (such as conjugate pad 20 in FIG. 8) serves to, amongother things, hold a detector reagent. In some embodiments, a detectorreagent may be applied externally, for example, from a developer bottle,in which case a lateral flow device need not contain a conjugate pad(see, for example, U.S. Pat. No. 4,740,468).

Detector reagent(s) contained in a conjugate pad is typically releasedinto solution upon application of the test sample. A conjugate pad maybe treated with various substances to influence release of the detectorreagent into solution. For example, the conjugate pad may be treatedwith PVA or PVP (0.5% to 2%) and/or Triton X-100 (0.5%). Other releaseagents include, without limitation, hydroxypropylmethyl cellulose, SDS,Brij and β-lactose. A mixture of two or more release agents may be usedin any given application. In the particular disclosed embodiment, thedetector reagent in conjugate pad 20 is gold-conjugated oxidizedcardiolipin, labeled Protein A, labeled Protein G, or labeled anti-humanIgG.

4. Absorbent Pad

The use of an absorbent pad 18 in a lateral flow device is optional. Theabsorbent pad acts to increase the total volume of sample that entersthe device. This increased volume can be useful, for example, to washaway unbound analyte from the membrane. Any of a variety of materials isuseful to prepare an absorbent pad, see, for example, Table 1. In somedevice embodiments, an absorbent pad can be paper (i.e., cellulosicfibers). One of skill in the art may select a paper absorbent pad on thebasis of, for example, its thickness, compressibility,manufacturability, and uniformity of bed volume. The volume uptake of anabsorbent made may be adjusted by changing the dimensions (usually thelength) of an absorbent pad.

D. Antigen-Coated Microtiter Plates

Other common solid-surface-based immunoassays are various forms ofimmunoabsorbent assay, such as enzyme-linked immunoabsorbent assay (orELISA). These assays typically involve antigen (e.g., oxidizedcardiolipin-attachment molecule complex) applied in the wells of amicrotiter plate. In this assay, a test sample (e.g., serum or blood)potentially containing an analyte of interest (e.g., anti-lipoidalantibody) is placed in the wells of a microtiter plate that contain animmobilized binding partner (e.g., oxidized cardiolipin-attachmentmolecule complex) specific for the subject analyte. The analytespecifically binds the immobilized antigen; then, unbound materials arewashed away leaving primarily the analyte-antigen complex bound to theplate. This complex can be detected in a variety of manners, as has beendescribed in detail above. One advantage of the microtiter plate formatis that multiple samples can be tested simultaneously (together withcontrols) each in one or more different wells of the same plate; thus,permitting high-throughput analysis of numerous samples.

E. Antigen Combinations

Each of the immunoassays and/or immunoassay devices discussed herein(e.g., ELISA, dipstick, flow-through device or lateral flow device) canbe, in some embodiments, formatted to detect multiple analytes by theaddition of capture reagents specific for the other analytes of interest(e.g., treponemal antigens). For example, certain wells of a microtiterplate can include capture reagents specific for the other analytes ofinterest. Some immunoassay device embodiments can include secondary,tertiary or more capture areas containing capture reagents specific forthe other analytes of interest.

Particular embodiments involve immunoassay devices that concurrentlydetect anti-lipoidal antibody and treponemes or anti-treponemalantibodies in fluid samples (such as, human serum). Such combinationdevices further include a treponemal capture area involving (a) animmobilized treponemal antigen capable of being specifically bound by ananti-T. pallidum antibody, or (b) an immobilized anti-T. pallidumantibody that specifically binds a mobile treponemal antigen. As usedherein, a “treponemal antigen” is an antigen containing at least oneantigenic determinant that specifically binds anti-T. pallidumantibodies. Numerous treponemal antigens have been described in the art;see, for example, U.S. Pat. Nos. 6,479,248; 6,248,331; 5,681,934;5,578,456; 4,868,118; and 4,740,467. For instance, polypeptides of atleast the following apparent molecular weights have been described as T.pallidum antigens: 16-20 kDa, 18 kDa, 18-23 kDa, 25 kDa, 35 kDa, 37 kDa,37-46 kDa, 38 kDa, 39 kDa, 41 kDa, 43 kDa, 44 kDa, 46 kDa, 47 kDa, 58kDa, 150 kDa; and 180 kDa (for more particular detail, see U.S. Pat. No.4,846,118).

Treponemal antigens and anti-T. pallidum antibodies are polypeptides;thus, when used as capture reagents, these molecules can be directlyadhered to a solid support (such as, nitrocellulose, nylon or PVDF).Nonetheless, it is contemplated that treponemal antigens or anti-T.pallidum antibodies can be immobilized (directly or indirectly) on asolid support by any available method.

A detector reagent can be used to detect the formation of a complexbetween a treponemal capture reagent and treponeme-specific analyte(such as, a treponeme, a treponemal antigen, or an anti-treponemalantibody). In some embodiments, a detector reagent (such as ananti-human Ab) can specifically detect a bound treponeme-specificanalyte (e.g., a human anti-treponemal antibody) and a boundanti-lipoidal antibody analyte (e.g., a human anti-lipoidal antibody).In other instances, two separate detector reagents for specificdetection of a bound treponeme-specific analyte (e.g., anti-treponemalantibody or treponemal antigen) or a bound anti-lipoidal antibodyanalyte are envisioned.

The operation of an immunoassay device useful for performing concurrenttreponemal and non-treponemal tests is substantially similar to devicesdescribed elsewhere in this specification. One particular feature of acombination device is that a fluid sample applied to a sampleapplication area is able to contact (e.g., flow to or flow through) eachof an anti-lipoidal antibody capture area and to a treponemal capturearea.

Other immunoassay and immunoassay devices embodiments involvecombinations of oxidized cardiolipin-containing antigens (e.g., oxidizedcardiolipin-attachment molecule complex) and other antigens specific foranti-lipoidal antibody; for example, immobilized lipoidal antigencomprising cardiolipin, lecithin and cholesterol. Immobilization oflipoidal antigen is described in detail in PCT/US2006/024117, which isincorporated herein in its entirety by reference. In one example, alipoidal antigen comprising cardiolipin (e.g., naturally occurring orsynthetic cardiolipin), lecithin and cholesterol is contacted with apopulation of Fab fragments specific for the lipoidal antigen to providea lipoidal antigen-Fab complex, which complex is readily attachable to asolid support in combination with oxidized cardiolipin-containingantigen.

VII. Kits

Disclosed herein are kits for use in detecting anti-lipoidal antibodiesin a sample (such as, a biological sample). Such kits can also be used,for example, in the diagnosis of diseases in which the presence ofanti-lipoidal antibodies is symptomatic of the disease (such as,syphilis or lupus). Certain embodiments of the disclosed kits aregenerally portable and provide a simple, rapid, and/or cost-effectiveway to detect anti-lipoidal antibodies and/or diagnose disease (such assyphilis) without the need for laboratory facilities, such as in apoint-of-care facility.

Kits include one or more immunoassay devices (and/or antigen-coatedmicrotiter plates) as disclosed herein and a carrier means, such as abox, a bag, a satchel, plastic carton (such as molded plastic or otherclear packaging), wrapper (such as, a sealed or sealable plastic, paper,or metallic wrapper), or other container. In some examples, kitcomponents will be enclosed in a single packaging unit, such as a box orother container, which packaging unit may have compartments into whichone or more components of the kit can be placed. In other examples, akit includes one or more containers, for instance vials, tubes, and thelike that can retain, for example, one or more biological samples to betested, positive and/or negative control samples or solutions (such as,a positive control serum containing anti-lipoidal or treponemalantibodies), diluents (such as, phosphate buffers, or saline buffers),detector reagents (e.g., for external application to a kit device),substrate reagents for visualization of detector reagent enzymes (suchas, 5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium indimethyl formamide), and/or wash solutions (such as, Tris buffers,saline buffer, or distilled water).

Other kit embodiments include syringes, finger-prick devices, alcoholswabs, gauze squares, cotton balls, bandages, latex gloves, incubationtrays with variable numbers of troughs, adhesive plate sealers, datareporting sheets, which may be useful for handling, collecting and/orprocessing a biological sample. Kits may also optionally containimplements useful for introducing samples into a sample chamber of animmunoassay device, including, for example, droppers, Dispo-pipettes,capillary tubes, rubber bulbs (e.g., for capillary tubes), and the like.Still other kit embodiments may include disposal means for discarding aused immunoassay device and/or other items used with the device (such aspatient samples, etc.). Such disposal means can include, withoutlimitation, containers that are capable of containing leakage fromdiscarded materials, such as plastic, metal or other impermeable bags,boxes or containers.

In some examples, a disclosed kit will include instructions for the useof an immunoassay device or antigen-coated plate. The instructions mayprovide direction on how to apply sample to the test device or plate,the amount of time necessary or advisable to wait for results todevelop, and details on how to read and interpret the results of thetest. Such instructions may also include standards, such as standardtables, graphs, or pictures for comparison of the results of a test.These standards may optionally include the information necessary toquantify analyte using the test device, such as a standard curverelating intensity of signal or number of signal lines to an amount ofanalyte therefore present in the sample.

EXAMPLES

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

Example 1 Oxidation of Cardiolipin

This example describes the oxidation of double bonds in the unsaturatedfatty acids of unmodified cardiolipin to carboxyl groups.

Approximately 100 mg of lyophilized cardiolipin (Avanti Polar Lipids(Alabaster, Ala.)) was dissolved in 2 mls of t-butanol and placed in avial under an argon atmosphere. Sixty (60) mg of sodium m-periodate(NaIO₄) was dissolved in 600 μl of distilled water and added dropwise tothe cardiolipin suspension with constant stirring. Promptly thereafter,8 mg of potassium permanganate (KMnO₄), dissolved in 400 μl of distilledwater, was added dropwise to the NaIO₄ reaction mixture with constantstirring. The mixture changed color (FIG. 1B) and was allowed to mix for24-48 hours at room temperature.

The degree of cardiolipin oxidation was qualitatively determined by thinlayer chromatography (TLC). Approximately 10 ml of achloroform:methanol:ammonium hydroxide solution (61.9:30.9:7.1 byvolume) was placed in a 100 ml beaker. A folded filter paper was placedinside the beaker and was allowed to become saturated with the solvent.

One (1) μl of the NaIO₄/KMnO₄ reaction mixture and 1 μl of a 20 mg/mlcardiolipin t-butanol (control) solution were placed 1 cm from the endof a 7.5 cm×2.5 cm strip of TLC silica gel. The strip was placed againstthe filter paper in the beaker, and the solvent was allowed to migrateto the top of the strip.

After removing the strip from the solvent, it was allowed to evaporateto dryness. Thereafter, the strip was wetted by brief submersion in 50ml of 5% ethanol containing at least 10 ml of molybdenum blue (SprayReagent; Alltech; Part No. 18213). The strip was heat dried with the aidof a hair dryer to develop the stain.

Upon completion of cardiolipin oxidation, the NaIO₄/KMnO₄ reactionmixture reaction was stopped by adding 80 mg of sodium bisulfite in 200μl in distilled water with constant stirring. The colored mixture thenturned colorless (compare FIG. 1B and FIG. 1C). The stopped reactionmixture was centrifuged at 1000×g for 5 minutes. After centrifugationthere were two visible phases (FIG. 1D).

Optionally, the supernatant t-butanol phase was placed in an evaporationround flask and dried under vacuum. Alternatively, the t-butanol phasemay be dialyzed against 10 mM phosphate buffer, pH. 8.0, or otherbuffer, to replace the alcohol solvent with a solution that is suitablefor the next-intended use of the oxidized cardiolipin contained therein,for example, for use in immunochromatographic assays, or, for example,for conjugation to an attachment molecule.

As shown in FIG. 2, unmodified cardiolipin migrates primarily to the topof a TLC strip, as indicated in lane A by the dark spot furthest fromthe point of origin. Cardiolipin naturally comprises a heterogeneouspopulation of molecules, as described in Section W. Because linoic acidmakes up approximately 90% of the fatty acid side chains in cardiolipin,predominantly linoic-acid-containing cardiolipin molecules (and closelyrelated cardiolipin forms) are likely represented by the dark spot inFIG. 2, lane A. Lesser represented cardiolipin forms likely havediffering mobilities and may account for the slight smear observed inlane A in the direction of migration.

As described previously, oxidation of cardiolipin oxidizes alkenes,cleaves the fatty acid side chains, and introduces carboxyl groups intoone or more of the fatty acid side chains. Carboxyl groups present inoxidized cardiolipin interact more strongly with the silica substrate ofa TLC strip, which retards the migration of oxidized cardiolipin formsalong the strip. Hence, in oxidized cardiolipin preparations (see FIG.2, lanes B and D1), the smear in the direction of migration becomes morepronounced (compare lane A with lanes B and D1). In addition, oxidativecleavage of the fatty acid side chains produces alkyl carboxylates,which have little mobility on a TLC strip under these conditions. Asshown in FIG. 2, lanes B and D2, the carboxylates are thought to berepresented by a white spot (due to dye exclusion) coextensive with thepoint of origin. Following cardiolipin oxidation as described in thisexample, oxidized forms of cardiolipin were found predominantly in thet-butanol phase (shown in FIG. 2, lane D1), while alkyl carboxylateswere found predominantly in the aqueous phase (see FIG. 2, lane D2).

As judged by reactivity with syphilitic serum (see, e.g., Example 4),the upper t-butanol phase (“alcohol phase”) (see FIG. 2, lane D1)contained oxidized cardiolipin species, while the lower aqueous phase(see FIG. 2, lane D2) was believed to contain mostly a mixture ofmalonic acid and caproic acid byproducts based on TLC results.

Example 2 Activation of Oxidized Cardiolipin and Conjugation withAttachment Molecules

This example demonstrates several methods of conjugating oxidizedcardiolipin with BSA or KLH using EDC and NHS.

A. Method One

After complete evaporation of the t-butanol phase described in Example1, the dry oxidized cardiolipin preparation was suspended in 2 ml ofN,N-formamide to a concentration of approximately 25 mg/ml. Ten (10) mgof EDC and 10 mg of NHS were dissolved in 1 ml of distilled water. TheEDC/NHS solution was then added to the formamide solution containingoxidized cardiolipin. The resultant mixture was stirred for 1 hour atroom temperature. As one of skill in the art will recognize, EDC and NHSwill convert carboxyl groups in oxidized cardiolipin to amine-reactiveNHS esters.

Up to a 40-fold molar excess of BSA or KLH was added to theEDC/NHS/oxidized cardiolipin mixture, and the mixture was stirred forone hour at room temperature. Preferably, about 1 ml of a 5 mg/ml KLH orBSA solution was added. As the amount of BSA or KLH in the reactionmixture is increased (e.g., to 10 mg/ml or greater), the more likely itbecomes that the protein component will crosslink to itself, which willcause a precipitate to appear. In this event, it may be necessary toseparate the precipitate by centrifugation and discard it.

The reaction mixture was clarified, as needed, and then dialyzed againsttwo, one-liter changes of 10 mM phosphate buffer, pH. 8.0. Thepreparation was concentrated by membrane filtration (Centricon filter;Millipore) to approximately 10 mg/ml oxidized cardiolipin. The oxidizedcardiolipin solution may be stored at 2-8° C. or −20° C. until needed.

B. Method Two

After complete evaporation of the t-butanol phase, as described inExample 1, the dry oxidized cardiolipin preparation was suspended in 2ml of N,N-formamide to a concentration of approximately 25 mg/ml. Thisformamide mixture was then dialyzed against two, one-liter changes of 10mM phosphate buffer, pH. 8.0 prior to the addition of the EDC/NHSsolution, as described in Method One in this example. The stepsfollowing addition of the EDC/NHS solution are the same as thosedescribed in Method One.

C. Method Three

An oxidized cardiolipin preparation was prepared by dialyzing thet-butanol phase described in Example 1 against 10 mM phosphate buffer,pH 8.0, followed by lyophilization to dryness. For use in a subsequentreaction, the lyophilized oxidized cardiolipin was reconstituted inwater to approximately 25 mg/ml. Then, 10 mg of EDC and 10 mg of NHSdissolved in 1 ml of distilled water was added to the oxidizedcardiolipin solution with stirring for 1 hour at room temperature. Thesteps following addition of the EDC/NHS solution are the same as thosedescribed in Method One.

Example 3 Biotinylation of Oxidized Cardiolipin

This example describes the biotinylation of oxidized cardiolipin.

Ten (10) mg of oxidized cardiolipin (see Example 1) was dissolved in oneml of 100 mM N-morpholinoethane sulfonic acid (MES). One hundred (100)μl of 20 mg/ml biotin-PEO-amine was added to the oxidized cardiolipinsolution. Immediately thereafter, 25 μl of a freshly prepared 1 mg/mlEDC solution was added to the biotin/cardiolipin mixture, and thesolution was mixed for 2 hours at room temperature. The reaction mixturewas then dialyzed against two changes of 10 mM phosphate buffer, pH 8.0.The biotinylated product was concentrated by lyophilization and canthereafter be reconstituted with distilled water to a desiredconcentration, for example, 10 mg/ml.

Example 4 Antigencity of Cardiolipin Preparations

This example demonstrates that oxidized cardiolipin as prepared inExample 1 retains antigenicity when tested against syphilitic serumusing two immunoassays.

A. RPR Inhibition Test

One hundred (100) μl of syphilitic serum (lot SOL) was mixed with 50 μlof the oxidized cardiolipin-BSA or -KLH preparations (approximately2.5-5 mg/ml) described in Table 2. If an oxidized cardiolipinpreparation retains antigenicity, anti-lipoidal antibodies present inthe serum will specifically bind to the oxidized cardiolipinpreparation, which will act to reduce the number of availableanti-lipoidal antibody binding sites. In other words, the serum will be“stripped” of some or all of the anti-lipoidal antibody binding sites.Serum that has been stripped of anti-lipoidal antibody binding siteswill be less reactive when tested in the conventional RPR test.

TABLE 2 Oxidized Cardiolipin Preparations Method of Cardiolipin Hrs.Conjugate Preparation Prep. No. Oxidation Type (see Example 2) 1 48Oxidized Method One cardiolipin-BSA 2 Duplicate of Preparation 1 3 48Oxidized Method One cardiolipin-KLH 4 Duplicate of Preparation 3 5 24Oxidized Method Three cardiolipin-BSA 6 24 Oxidized Method Threecardiolipin-KLH 7 24 Oxidized Method One cardiolipin-BSA 8 Duplicate ofPreparation 7. 9 24 Oxidized Method One cardiolipin-KLH 10 Duplicate ofPreparation 9 11 24 Oxidized Method Two cardiolipin-BSA conjugate 12 24Oxidized Method Two cardiolipin-KLH

The stripped (and control) serum was tested in an RPR test in accordancewith Chapter 10 of the Manual of Tests for Syphilis, 9th Edition,Washington, D.C.: American Public Health Association, 1998.

As shown in Table 3, control syphilitic serum, to which 50 μl of 5 mg/mlunconjugated BSA was added, reacted with the RPR test antigen at a 1:8serum dilution. Incubation of the syphilitic serum with each of thecardiolipin preparations 1-12 (described in Table 2) inhibited thereactivity of the serum with the RPR test antigen by at least two-fold.This result indicates that each of the cardiolipin preparations reactedto some extent with the anti-lipoidal antibodies present in the testsyphilitic serum, and thereby inhibited the stripped serum from reactingwith the RPT test antigen. For example, preincubation of the syphilitictest serum with cardiolipin preparations 2, 11, and 12 completelyinhibited the reactivity of the serum in the RPR test.

TABLE 3 RPR Inhibition Test Results Cardiolipin Preparation* Used forSerum Serum (Lot 50 L) Dilution Preincubation 1:1 1:2 1:4 1:8 1:16 1:32None (i.e., Control) R R R₍₋₎ Rm N N Preparation 1 Rm N N N N NPreparation 2 N N N N N N Preparation 3 Rm₍₋₎ N N N N N Preparation 4Rm₍₋₎ N N N N N Preparation 5 Rm₍₋₎ N N N N N Preparation 6 Rm₍₋₎ N N NN N Preparation 7 Rm₍₋₎ N N N N N Preparation 8 Rm Rm₍₋₎ N N N NPreparation 9 Rm Rm₍₋₎ N N N N Preparation 10 Rm Rm₍₋₎ N N N NPreparation 11 N N N N N N Preparation 12 N N N N N N *See Table 2 fordescription of cardiolipin preparations R = strong positive reactionR₍₋₎ = positive reaction Rm = weak reaction Rm₍₋₎ = very weak reaction N= negative reactionB. Immuno-Blot Test

Dot blot assays were performed using the Immun-Blot® Assay Kit (BioRad)with a goat anti-human IgG alkaline-phosphatase conjugate in accordancewith the manufacturer's instructions. As shown in FIG. 5, oxidizedcardiolipin-BSA preparation 5 (see Table 2) reacted with syphiliticserum (Lot 00) over a wide range of antigen (7.5-120 μg) and antibody(1:10-1:640 dilution) concentrations. In comparison, preparation 5 didnot react at all with normal, human serum (i.e., non-syphilitic serum)over the same ranges of antigen and antibody concentrations.

FIG. 5 also shows that serum Lot 00 (1:640 dilution) reacts with a wholecell T. pallidum preparation, which confirms that the serum donor hadbeen infected with T. pallidum.

Example 5 Preparation of Oxidized Cardiolipin Gold Conjugate

This example demonstrates the conjugation of oxidized cardiolipin-BSA or-KLH with colloidal gold, and determination of useful conditions for thepreparation of such gold conjugates. This colloidal gold preparation canbe used as the conjugate in a lateral flow strip, such as the lateralflow strip described in association with FIG. 7.

A. Determination of a Useful pH for Oxidized Cardiolipin/Colloidal GoldConjugation Reaction

Approximately 25 ml of 10 mM phosphate buffer was placed in a 50-mlbeaker, and adjusted to pH 5.0 with 0.2 M phosphoric acid. Two 0.5-mlaliquots of the buffer at pH 5.0 were transferred to two 12×75 mm testtubes, one labeled “test” and the other labeled “control.” Then, the pHof the phosphate buffer remaining in the beaker was sequentiallyadjusted to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 using0.2 M potassium carbonate. At each pH, two 0.5-ml aliquots weretransferred to a “test” and “control” test tube as described for the pH5.0 sample.

Six (6) μL of a 5 mg/ml cardiolipin-BSA preparation or a 5 mg/mlcardiolipin-KLH preparation (30 μg), prepared as described in Example 2,was added to each of the “test” and “control” tubes, and mixed well.

Approximately 25 ml of 40-nm colloidal gold (1% solution) (BritishBiocell International, London, England) was placed in a separate 50-mlbeaker, and a series of “control” and “test” colloidal gold samples atpH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 wereproduced as described above.

One (1) ml of colloidal gold at each pH was added to the “test” and“control” cardiolipin solutions having the corresponding pH. Thegold/cardiolipin solutions were mixed well, and incubated for 20 minutesat room temperature. Then, 200 μL 2 M NaCl was added to the set of tubeslabeled “test” and 200 μL of distilled water was added to the set oftubes labeled “control.” The contents of both sets of tubes were allowedto incubate for 30 minutes at room temperature.

The optical density at 580 nm (OD₅₈₀) of each “test” sample was readagainst the corresponding “control” sample. The pH of the sample withthe lowest OD₅₈₀ was determined to be a favorable pH for formation of agold conjugate of the oxidized cardiolipin-BSA or -KLH preparation.

Colloidal gold particles have a negatively charged surface due to thelayer of negative ions adsorbed onto the gold particle surface duringthe manufacturing process. Proteins, such as BSA or KLH in oxidizedcardiolipin-BSA or -KLH preparations, will be attracted to negativelycharged gold particles through ionic, hydrophobic, and dativeinteractions. These interactions underlie the formation of colloidalgold-protein conjugates. At the pI of a protein conjugated to a goldparticle (i.e., the pH where the protein has a net zero charge), theconjugate will be the most stable.

The addition of NaCl to unconjugated colloidal gold particles willdisrupt the layer of negatively charged ions adsorbed to the gold'ssurface. As a result, the gold particle will dissociate and ultimatelyrelease gold ions (i.e., Au⁺) into solution. The free gold ions may bemeasured at OD₅₈₀. In contrast, a protein-gold conjugate (e.g., oxidizedcardiolipin-BSA-gold conjugate or oxidized cardiolipin-KLH-goldconjugate) is resistant to disruption by NaCl at the pI of the proteincomponent of the conjugate (e.g., the pI of oxidized cardiolipin-BSA orthe pI of oxidized cardiolipin-KLH). Hence, in this example, the pH ofthe sample with the lowest OD₅₈₀ is selected.

Because the molar ratio of cardiolipin to attachment molecule in eachoxidized cardiolipin-attachment molecule (e.g., oxidized cardiolipin-BSAor oxidized cardiolipin-KLH) preparation may differ, the pI of each suchpreparation may also differ. Thus, it is beneficial to determine theuseful pH, as described in this example, for each oxidizedcardiolipin-attachment molecule preparation made.

B. Determination of a Useful Cardiolipin-BSA or Cardiolipin-KLHConcentration for a Colloidal Gold Conjugation Reaction

One hundred (100) μl of distilled water was added to each in a series ofeleven 12×75 mm test tubes. Then, 1 μl, 2 μl, 5 μl, 7 μl, 10 μl, 15 μl,25 μl, 50 μl, 75 μl or 100 μl of a 1 mg/ml solution of eithercardiolipin-BSA or cardiolipin-KLH was added to a corresponding tube inthe series. The eleventh tube in the series served as a control. Eachtube was mixed well, and allowed to incubate at room temperature for 5minutes. At this point, the solution in each tube was red in color. Fivehundred (500) μl of a 10% NaCl solution was then added to each tube withshaking, and the tubes were again incubated at room temperature for 5minutes. The color of the solution of each tube was observed by eye withsome turning from red to blue after the addition of NaCl. The minimumamount of cardiolipin-attachment molecule useful for stabilizing thegold conjugate was determined to be the lowest concentration ofcardiolipin-attachment molecule in a blue-colored solution after NaCladdition.

Higher concentrations of oxidized cardiolipin-attachment moleculecomplex, though useful, are less preferred because excess oxidizedcardiolipin-attachment molecule complexes may form weaker associationswith the gold particles, for example, by layering upon a layer ofcardiolipin-attachment molecule complexes that previously associatedwith the gold particles.

Similarly, lower concentrations of oxidized cardiolipin-attachmentmolecule complex may be useful, but are less preferred becauseunconjugated gold particles may provide background “noise” in otherapplications of the gold-conjugated cardiolipin-attachment complexes.Alternatively, unconjugated gold particles may be separated fromgold-conjugated cardiolipin-attachment molecule complexes using methodscommonly known in the art, such as centrifugation or filtration.

C. Cardiolipin-BSA or Cardiolipin-KLH Gold-Conjugate Minipreps.

For a particular oxidized cardiolipin-BSA or -KLH preparation, determinea useful pH and a useful oxidized cardiolipin-BSA or oxidizedcardiolipin-KLH concentration as described above. If the useful pH is pH8.0 or higher, then add 5 ml of 10 mM borate buffer to the amount oflyophilized oxidized cardiolipin-attachment molecule complex necessaryto achieve the useful concentration; then, adjust the pH to the usefulpH. If the useful pH is pH 8.0 or lower, add 5 ml of 10 mM phosphatebuffer to the amount of lyophilized oxidized cardiolipin-attachmentmolecule complex necessary to achieve the useful concentration; then,adjust the pH to the useful pH. Next, add 10 ml of 40-nm colloidal gold(1% solution adjusted to the useful pH), and mix well. Incubate themixture for 20 minutes at room temperature. Then, add 1.6 ml of 10% BSAto a final concentration of 1% BSA, and incubate for an additional 20minutes at room temperature. Centrifuge the reaction mixture at 6500×gfor 10 minutes, and remove the supernatant. Resuspend the pellet in 0.5ml of resuspension buffer (150 mM NaCl, 20 mM Trizma base, 10% sucrose,5% Trehalose, 0.1% BSA, 0.05% sodium azide). The resuspended pelletcontaining gold conjugated oxidized cardiolipin-BSA or -KLH may be usedfor a variety of purposes, including without limitation as a detectorreagent for anti-lipoidal antibodies in a lateral flow device.

Example 6 Attachment of Cardiolipin-Attachment Molecule Complexes toNitrocellulose

This example describes a representative method for attachingcardiolipin-protein complexes to a solid surface, in this case,nitrocellulose.

For a particular oxidized cardiolipin-BSA or -KLH preparation, determinea useful pH as described previously. Resuspend lyophilized oxidizedcardiolipin-BSA or -KLH complex in either 10 mM sodium acetate buffer(for useful pH values between 4.0-5.6) or 10 mM phosphate buffer (foruseful pH values between 7.0-9.0). Adjust the solution to the useful pH,with 2 M acetic acid for pH values between 4.0-5.6 or with 1 M mono- ordi-sodium phosphate for pH values between 7.0-9.0. Ethanol (0.5%) mayoptionally be added to the oxidized cardiolipin solution to improvereagent application by lowering the viscosity of the solution. Then,apply the oxidized cardiolipin-BSA or -KLH solution to nitrocelluloseusing a Matrix 1600 reagent dispensing module (Kinematic Automation,Twain Harte, Calif.) in accordance with the manufacturer's directions.

After application of the oxidized cardiolipin-BSA or -KLH preparation tonitrocellulose, the membrane should be dried for 30 minutes at 37° C.followed by 2 hours in a vacuum dessicator prior to use, for example, ina lateral flow device.

Example 7 Detection of Anti-Lipoidal Antibodies in Human Serum with anOxidized Cardiolipin-Protein Conjugate Capture Reagent

This example demonstrates that anti-lipoidal antibodies in syphiliticserum may be detected using oxidized cardiolipin-protein conjugatecapture reagent, which is immobilized in a nitrocellulose membrane, inconcert with a mobile oxidized cardiolipin-protein-gold conjugatedetector reagent.

One (1) μl of preparations 1-17 (as described in Table 2) were eachapplied to separate nitrocellulose membranes as described in Example 6,and set aside. A colloidal gold conjugate was prepared as described inExample 5 using oxidized cardiolipin preparation 6 (see Table 2).

Syphilitic serum (Lot 00) or non-reactive (normal, human) serum wasdiluted 1:10 and placed in an appropriate number of separate wells of a40-well microtitre plate. Three (3) μl of gold-conjugated preparation 6(for use as a detector reagent) was then added to each well.Nitrocellulose strips containing each of the immobilized preparations 1through 17 (see Table 2) were then placed into wells containing theantibody and detector reagent solutions. The solution in the wellsflowed up the strip by capillary action. Each immobilized oxidizedcardiolipin preparation was tested against both syphilitic andnon-reactive (i.e., control) serum.

As shown in Table 4, many of the oxidized cardiolipin preparationsimmobilized in or on nitrocellulose showed a positive reaction, which isindicative of a “sandwich” complex between the immobilized oxidizedcardiolipin capture reagent, at least one anti-lipoidal antibody, andthe oxidized cardiolipin gold conjugate detector reagent. Reactivity wasmeasured on a scale of 0 (Neg) to 4+ with 4+ indicating the strongestsignal observed on the nitrocellulose strip.

TABLE 4 Cardiolipin Reactive Serum Preparation Lot 00 Non-Reactive Serum1 2+ Neg 2 +/− +/− 3 1+ Neg 4 1+ Neg 5 4+ Neg 6 2+ Neg 7 1+ +/− 8 1+ Neg9 +/− +/− 10 +/− +/− 11 2+ Neg 12 1+ Neg

Example 8 Oxidation of Mixtures of Cardiolipin and Lecithin

This example describes the oxidation of mixtures of cardiolipin andlecithin, and demonstrates that oxidized mixtures of cardiolipin andlecithin perform at least as well as oxidized cardiolipin alone in theassays described in Examples 4-7.

Cardiolipin and lecithin were mixed together in ratios of 1:1, 1:3, and1:5 cardiolipin to lecithin by weight. The mixtures were then oxidizedas described in Example 1. As shown in FIG. 8, lane A, a mixture ofunmodified cardiolipin and unmodified lecithin resolves into two spotson a TLC strip. Unmodified cardiolipin migrates to the top of the TLCstrip, as indicated by the dark spot furthest from the point of origin(see also, FIG. 2, lane A). Unmodified lecithin migrates somewhat moreslowly on the TLC place and is shown as the dark spot approximatelyhalfway between the cardiolipin and the point of origin.

As described previously, oxidation under the conditions of Example 1oxidizes alkenes, cleaves the fatty acid side chains, and introducescarboxyl groups into one or more of the fatty acid side chains ofcardiolipin and lecithin. Carboxyl groups present in oxidized moleculesinteract more strongly with the silica substrate of a TLC strip, whichretards the migration of the oxidized forms along the strip. Hence, inoxidized mixtures of cardiolipin and lecithin, a pronounced smear in thedirection of migration is observed (see FIG. 8, lanes B and C). Inaddition, oxidative cleavage of the fatty acid side chains producesalkyl carboxylates, which have little mobility on a TLC strip underthese conditions. As especially evident in FIG. 8, lane D, thecarboxylates are thought to be represented by a white spot (due to dyeexclusion), which is coextensive with the point of origin. Followingoxidation of cardiolipin/lecithin mixtures as described in this example,oxidized forms of cardiolipin were found predominantly in the t-butanolphase (shown in FIG. 8, lane C), while alkyl carboxylates were foundpredominantly in the aqueous phase (see FIG. 8, lane D).

A. BSA and KLH Conjugation of Oxidized Cardiolipin/Lecithin Mixtures

Oxidized mixtures of cardiolipin and lecithin were conjugated to eitherBSA or KLH, as described in Method 3 of Example 2. The BSA- orKLH-conjugated cardiolipin/lecithin preparations are described in Table5.

TABLE 5 Cardiolipin/Lecithin Mixtures Amount of Attachment Cardiolipin(CL) CL:L Ratio Attachment Molecule Per Lecithin (L) Prep. No. (byweight) Molecule Reaction (mg) C/L 1 1:1 BSA 5 C/L 2 1:1 BSA 2 C/L 3 1:1KLH 2 C/L 4 1:1 KLH 1 C/L 5 1:3 BSA 5 C/L 6 1:3 BSA 2 C/L 7 1:3 KLH 2C/L 8 1:3 KLH 1 C/L 9 1:5 BSA 5 C/L 10 1:5 BSA 2 C/L 11 1:5 KLH 2 C/L 121:5 KLH 1B. RPR Inhibition Test of Oxidized Cardiolipin/Lecithin Mixtures

The antigenicities of the cardiolipin/lecithin preparations described inTable 5 were tested in the RPR Inhibition Test, as described in Example3. As shown in Table 6, each of the cardiolipin/lecithin preparationscompletely inhibited the reactivity of syphilitic serum in thetraditional RPR test.

TABLE 6 RPR Inhibition Test Results for Cardiolipin/Lecithin MixturesCardiolipin/Lecithin Prep. Used for Serum Serum (Lot 50 L) DilutionPreincubation 1:1 1:2 1:4 1:8 1:16 1:32 None (i.e., Control) R R R₍₋₎ RmN N C/L 1 N N N N N N C/L 2 N N N N N N C/L 3 N N N N N N C/L 4 N N N NN N C/L 5 N N N N N N C/L 6 N N N N N N C/L 7 N N N N N N C/L 8 N N N NN N C/L 9 N N N N N N C/L 10 N N N N N N C/L 11 N N N N N N C/L 12 N N NN N N R = strong positive reaction R₍₋₎ = positive reaction Rm = weakreaction Rm₍₋₎ = very weak reaction N = negative reaction

This result demonstrates that the oxidized cardiolipin/lecithin mixturesvery efficiently strip (i.e., remove) anti-lipoidal antibodies from thesyphilitic serum, so that such serum is non-reactive in the traditionalRPR test.

C. Immunodot Testing of Oxidized Cardiolipin/Lecithin Mixtures

Dot blot assays were performed using the Immun-Blots Assay Kit (BioRad)with a goat anti-human IgG alkaline-phosphatase conjugate in accordancewith the manufacturer's instructions. As shown in FIG. 10, oxidizedcardiolipin/lecithin mixtures in the range 375 ng to 21 μg per dotreacted with syphilitic serum (Lot 94265; 1:50 dilution). In comparison,none of the tested amounts of the oxidized cardiolipin/lecithinpreparations showed any significant reaction with normal, human serum(i.e., non-syphilitic serum).

The preparations where cardiolipin and lecithin were oxidized in a 1:1ratio (by weight) and then conjugated to either BSA or KLH (i.e., 1:1BSA and 1:1 KLH) were most reactive with syphilitic serum. As little as375 ng of the 1:1 BSA preparation and 656 ng of the 1:1 KLH preparationbound to human antibodies present in the syphilitic serum in this assay.

Example 9 Detection of Anti-Lipoidal Antibodies in Human Serum withProtein-A or Anti-Human Antibody Capture Reagent

This example demonstrates that anti-lipoidal antibodies present in humanserum may be detected using protein-A or anti-human antibody capturereagent, which is immobilized in a nitrocellulose membrane, in concertwith a mobile oxidized cardiolipin-protein-gold conjugate detectorreagent.

Whole blood will be collected from a human subject thought to be at riskfor a condition that gives rise to serum levels of anti-lipoidalantibodies. Such conditions include, for example, T. pallidum infection(i.e., syphilis) or lupus. Serum will be separated from the whole bloodby methods well known in the art. The serum may be diluted, for example,with normal saline or other solutions that will not denature orotherwise affect the specific binding of anti-lipoidal antibodiespresent in the serum or otherwise interfere with the described method.

The serum sample will be applied to a nitrocellulose strip containing amobile or mobilizable, labeled oxidized cardiolipin-protein conjugatedetector reagent. Such conjugate may be, for example, gold-labeledoxidized cardiolipin-BSA, or gold-labeled oxidized cardiolipin-KLHconjugate, or a gold-labeled, BSA-conjugated mixture of oxidizedcardiolipin and oxidized lecithin, or a gold-labeled, KLH-conjugatedmixture of oxidized cardiolipin and oxidized lecithin.

Anti-lipoidal antibodies present in the serum sample will bind to thecardiolipin of the detector reagent and flow (such as by capillaryaction, or by lateral flow forces) to a portion of the nitrocellulosestrip where a protein-A or anti-human antibody capture reagent isimmobilized. The anti-lipoidal antibody complexed with the cardiolipindetector reagent will be captured by the protein A or anti-humanantibody capture reagent. Accumulation of detector reagent complexes inthis manner will result in a detectable signal (for example, a visiblesignal) on the nitrocellulose in the area where the capture reagent isimmobilized.

The appearance of a detectable signal as described will indicate thatanti-lipoidal antibodies are present in the serum sample, and may beuseful in the diagnosis of certain conditions, such as syphilis orlupus.

Example 10 Detection of Anti-Lipoidal Antibodies in Human Serum withEnzyme-Linked Immunoassay

This example demonstrates that anti-lipoidal antibodies present in humanserum may be detected by using oxidized cardiolipin conjugated to BSA orKLH as a capture reagent, which is immobilized in a microtiter plate forthe performance of an enzyme-linked immunoassay. Alternatives to BSA andKLH include synthetic protein MAPS, IgY, streptavidin, and avidin. Thewells of a 96-well microtiter plate were coated with 100 μl (10 μg/ml)of a solution containing 10 μg/ml oxidized cardiolipin-BSA or oxidizedcardiolipin-KLH complex. The solution was allowed to dry overnight at37° C. The wells were then blocked for 2 hours at room temperature withat least 200 μl 1% casein in Tris phosphate buffered saline (TPBS) pH7.2. The wells were washed once with 200 μl of TPBS. Then, 100 μl ofhuman serum (either control or from T. pallidum-infected individual)diluted 1:20, 1:40, 1:80 or 1:160 in 1% casein TPBS was added to eachwell. The microtiter plate was incubated at room temperature for 60minutes; followed by three washes with 200 μl TBST. One hundred (100) μlof goat anti-human antibody conjugated to horseradish peroxidase (HRP)diluted 1:3000 in 1% casein TPBS was added to each well at roomtemperature for 45 minutes. The microtiter plate was washed three timeswith TPBS prior to the addition of 100 μl per well of TMB substrate. Inthe presence of HRP, the TMB substrate changes color. The HRPenzyme-substrate reaction was stopped by the addition of 2M sulfuricacid. The solution in each well was read spectrophotometrically at 450nm.

As shown in Tables 7 and 8, anti-lipoidal antibodies present in humansyphilitic sera (“Reactive”) bound to oxidized cardiolipin-BSA or -KLHcomplexes immobilized in the wells of the microtiter plate. However,there was no significant binding of control (“Nonreactive”) serum to thesame immobilized antigen.

TABLE 7 Enzyme-Linked Immunoassay with Oxidized Cardiolipin-BSA AntigenSerum dilution Reactive Reactive Nonreactive Nonreactive 1:20 1.9381.712 0.103 0.066 1:40 0.569 0.769 0.004 0.005 1:80 0.348 0.495 0.0180.020 No sera 0.032 0.031 0.029 0.033 Duplicate tests (results shown)were performed for each data point. The syphilitic serum used for thisdata set has a titer of 1:64 as determined by the RPR test.

TABLE 8 Enzyme-Linked Immunoassay with Oxidized Cardiolipin-BSA AntigenSerum dilution Reactive Reactive Nonreactive Nonreactive 1:20 1.9701.700 0.071 0.062 1:40 0.825 0.842 0.045 0.039 1:80 0.552 0.479 0.0310.027  1:160 0.281 0.245 0.022 0.022 No sera 0.046 0.034 0.021 0.019Duplicate tests (results shown) were performed for each data point. Thesyphilitic serum used for this data set has a titer of 1:128 asdetermined by the RPR test.

While this disclosure has been described with an emphasis uponparticular embodiments, it will be obvious to those of ordinary skill inthe art that variations of the particular embodiments may be used and itis intended that the disclosure may be practiced otherwise than asspecifically described herein. Features, characteristics, compounds,chemical moieties, or examples described in conjunction with aparticular aspect, embodiment, or example of the invention are to beunderstood to be applicable to any other aspect, embodiment, or exampleof the invention. Accordingly, this disclosure includes allmodifications encompassed within the spirit and scope of the disclosureas defined by the following claims.

1. A method for preparing oxidized cardiolipin that is immunoreactivewith anti-lipoidal antibodies, and capable of linking to a protein forattachment to a substrate, wherein the cardiolipin comprises a centralglycerol moiety and fatty acid side chains, the method comprising:reacting the cardiolipin with a periodate salt and a permanganate saltto oxidize fatty acid side chains of the cardiolipin to provide terminalcarboxyl groups on one or more of the side chains; adding a reducingagent to the cardiolipin suspension after reacting the cardiolipin withthe periodate salt and the permanganate salt to quench oxidation of thecardiolipin and to reduce a β-ketone formed in the central glycerolmoeity to a β-hydroxyl group so as to retain immunogenicity of thecentral glycerol moiety; and activating the carboxyl groups of theoxidized cardiolipin to covalently attach a protein carrier to at leastone of the carboxyl groups.
 2. The method of claim 1, wherein reactingthe cardiolipin occurs in an alcohol solvent or in an argon atmosphere.3. The method of claim 2, wherein the alcohol is one or more oft-butanol, ethanol, propanol, or methanol.
 4. The method of claim 1,wherein activating the carboxyl groups comprises reacting the oxidizedcardiolipin with a carbodiimide.
 5. The method of claim 4, wherein thecarbodiimide comprises 1-ethyl-3-(3-dimethylamino propyl)carbodiimide(EDC).
 6. The method of claim 1, wherein the cardiolipin is reacted withthe periodate salt before the cardiolipin is reacted with thepermanganate salt.
 7. The method of claim 1, wherein the periodate saltis sodium m-periodate and the permanganate salt is potassiumpermanganate.
 8. The method of claim 7, wherein the molar ratio ofsodium m-periodate to cardiolipin is about 4:1 to about 5:1.
 9. Themethod of claim 7, wherein the molar ratio of potassium permanganate tocardiolipin is about 0.5:1 to about 1:1.
 10. The method of claim 1,wherein the reducing agent is sodium bisulfite.
 11. The method of claim1, further comprising: separating an aqueous and alcohol phase after theaddition of the reducing agent, and recovering oxidized cardiolipin fromthe alcohol phase; or covalently attaching a protein to at least one ofthe activated carboxyl groups to provide a cardiolipin-proteinconjugate.
 12. The method of claim 11, wherein covalently attaching aprotein comprises attaching bovine serum albumin (BSA), keyhole limpethemocyanin (KLH), or avidin.
 13. The method of claim 4, whereinactivating carboxyl groups further comprises reacting the carboxylgroups with an N-hydroxysuccinimide (NHS) after reacting the carboxylgroups with the EDC.
 14. The method of claim 11, wherein covalentlyattaching a protein comprises activating carboxyl groups by reacting theoxidized cardiolipin with a carbodiimide and then an NHS to provide aproduct, then conjugating the product with the protein.
 15. The methodof claim 14, wherein the protein comprises bovine serum albumin (BSA),keyhole limpet hemocyanin (KLH), or avidin.
 16. A method for preparingoxidized cardiolipin that is immunoreactive with anti-lipoidalantibodies, and capable of linking to a protein for attachment to asubstrate, wherein the cardiolipin comprises a central glycerol moietyand fatty acid side chains, the method comprising: reacting thecardiolipin with sodium m-periodate and potassium permanganate in at-butanol solvent under an argon atmosphere to oxidize the cardiolipinand provide terminal carboxyl groups on one or more of the fatty acidside chains; adding sodium bisulfite in aqueous solution to thecardiolipin suspension after reacting the cardiolipin with the sodiumm-periodate and the potassium permanganate to quench oxidation of thecardiolipin and to reduce a β-ketone formed in the central glycerolmoeity to a β-hydroxyl group so as to retain immunogenicity of thecentral glycerol moiety; and activating the terminal carboxyl groups ofthe oxidized cardiolipin to covalently attach a protein carrier to atleast one of the carboxyl groups, wherein activating the carboxyl groupscomprises reacting the oxidized cardiolipin with EDC and then NHS; andconjugating the protein to one or more of the activated carboxyl groups.